Tumor specific monoclonal antibodies

ABSTRACT

The invention provides tumor-specific human monoclonal antibodies and functional fragments. Also provided are nucleic acids encoding tumor-specific human monoclonal antibodies and functional fragments. A method for reducing neoplastic cell proliferation is also provided. The method consists of administering an effective amount of a tumor-specific human monoclonal antibody or functional fragment. Also provided is a method of detecting a neoplastic cell in a sample. The method consists of contacting a cell with a tumor-specific monoclonal antibody or functional fragment and detecting the specific binding of the human monoclonal antibody or functional fragment to the sample.

This application claims benefit of the filing date of U.S. ProvisionalApplication No. 60/______ (yet to be assigned), filed Nov. 19, 2001,which was converted from U.S. Ser. No. 09/989,901, and which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to cancer and, morespecifically, to human monoclonal antibodies for the treatment anddiagnosis of cancer.

Cancer is one of the leading causes of death in the United States. Eachyear, more than half a million Americans die, from cancer, and more thanone million are newly diagnosed with the disease. In cancer; neoplasticcells escape from their normal growth regulatory mechanisms andproliferate in an uncontrolled fashion, leading to the development of amalignant tumor. Tumor cells can metastasize to secondary sites iftreatment of the primary tumor is either not complete or not initiatedbefore substantial progression of the disease. Early diagnosis andeffective treatment of malignant tumors is therefore essential forsurvival.

The current methods for treating cancer include surgery, radiationtherapy and chemotherapy. A major problem with each of these treatmentsis their lack of specificity for cancer cells. For instance, surgicalremoval of the tumor is often incomplete. Even a few residual neoplasticcells can be lethal, as they can rapidly proliferate and metastasize toother sites. Radiation and chemotherapy also have serious limitations.These therapies target all growing cells of the body, including bothnormal and neoplastic cells. Due to their toxicity to normal tissues,the amount of radiation or chemotherapeutic agent that can be safelyused is often inadequate to kill all neoplastic cells. Additionally,their toxicity to normal tissues is manifested by unpleasant sideeffects, including nausea and hair loss, that severely reduce thequality of life for the cancer patient undergoing these treatments.Clearly, a more selective and effective means of treating cancer isneeded.

Monoclonal antibodies are homogeneous preparations of immunoglobulinproteins that specifically recognize and bind to regions, or epitopes,of their corresponding antigens. Neoplastic cells selectively expressantigens which are not present on normal cells. Thus, monoclonalantibodies can be produced that are directed against tumor-specificantigens. Such tumor-specific antigens can be linked to therapeuticmoieties that kill or arrest the growth of neoplastic cells. Inaddition, monoclonal antibodies can be linked to diagnostic moietiesthat allow the imaging of neoplastic cells. Thus, monoclonal antibodiesdirected against antigens selectively expressed by tumor cells comparedto normal cells can be beneficially used for the early detection andeffective treatment of cancer.

Most current immunotherapeutic strategies for cancer have been oflimited utility due to their reliance on mouse monoclonal antibodies.Mouse monoclonal antibodies can be produced easily and in virtuallyunlimited quantities using hybridoma technology. However, whenadministered to humans, they can be recognized as foreign by the humanimmune system and be neutralized before exerting their therapeuticeffect on the diseased tissue. Furthermore, the murine immune systemoften preferentially recognizes immunodominant epitopes of normal humanantigens present on tumor cells. Thus, human tumor-specific antigensoften fail to generate therapeutically beneficial murine antibodies.

Human monoclonal antibodies can overcome both of these limitations. Mostimportantly, human monoclonal antibodies are not as immunogenic asmurine antibodies. Therefore, tumor-specific human monoclonal antibodieswill be able to more effectively target and eliminate neoplastic cells.Furthermore, the human immune system is less likely to generate antigensagainst epitopes present on normal cells, increasing the odds ofgenerating and successfully identifying tumor-specific antigens.Additionally, the repertoire of the human immune system is differentfrom that of the mouse, containing potentially novel antibodyspecificities.

Current procedures to produce tumor-specific human monoclonal antibodieshave generally started with lymphocytes obtained from tumor-bearingpatients. These procedures rely on the stimulation and expansion oftumor-reactive lymphoctyes in vivo. These procedures are seriouslylimited by the narrow range of antigen specificities of activatedB-cells of cancer patients. As it is clearly not possible to immunizeindividuals in vivo with tumor cells, as one can with mice, it has notbeen possible to readily generate tumor-specific human monoclonalantibodies to any given antigen or tumor cell type. Procedures forgenerating tumor-specific antibodies of any desired specificity would bevery beneficial for effective immunotherapy and immunodiagnosis.

Thus, there exists a need for improved tumor-specific human monoclonalantibodies for the therapy and diagnosis of cancer. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides an isolated human monoclonal antibody orfunctional fragment thereof, including a complementary determiningregion having substantially the amino acid sequence of a CDR selectedfrom the group consisting of SEQ ID NOs:10, 12, 14, 16, 18, 20, 22, 24,26., 28, 30, 32, 34, 36, 38, 40, 42 and 44, wherein the human monoclonalantibody or functional fragment thereof specifically binds a neoplasticcell or antigen thereof.

The invention also provides an isolated nucleic acid, encoding a humanmonoclonal antibody or functional fragment thereof, including anucleotide sequence encoding substantially the amino acid sequence of aCDR encoded by a nucleotide sequence selected from the group consistingof SEQ ID NOs:9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43 and 45.

A method for reducing neoplastic cell proliferation is also provided.The, method consists of administering an effective amount of a humanmonoclonal antibody or functional fragment of the invention. Alsoprovided is a method of detecting a neoplastic cell in a sample. Themethod consists of contacting a cell with a monoclonal antibody orfunctional fragment of the invention and detecting the specific bindingof the human monoclonal antibody or functional fragment to the sample.The presence or increased level compared to a normal cell of themonoclonal antibody or functional fragment indicates the presence of aneoplastic cell in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the binding of LH11238 and LH13 antibodies to the surfaceof live H3464 cells. FIG. 1A shows fluorescent activated cell sorting(FACS) analysis using LH11238 antibody. FIG. 1B shows FACS analysisusing LH13 antibody.

FIG. 2 shows that LH13 antigen is secreted by H3396 cells. FIG. 2A showsthat conditioned medium from H3396 cells (closed circles) competes forbinding of LH13 antibody to fixed cell monolayers. FIG. 2B shows thatLH13 antigen is secreted by H3396 cells and binds to culture dishes.

FIG. 3 shows the purification of LH13 antigen by anion exchangechromatography on a Q Sepharose column.

FIG. 4 shows that LH13 antigen is susceptible to trypsin andendoglycosidase-F/peptide-N-lycosidase F treatment.

FIG. 5 shows characterization of recombinant LH13, LH11238 and variantFabs in an ELISA format. Panel A shows results for LH13 antibody (opencircles), the HCDR3 variant S97N (filled circles), the LCDR3 variantR91Y (open squares), and the combinatorial mutant 4H7 (filled squares)each titrated against partially enriched LH13 antigen and detected withgoat anti-human kappa alkaline phosphatase conjugate. Panel B showsresults for the LH11238 antibody (open circles) and the LCDR3 mutantsQ89W (open squares), N93C (open triangles), N94C (filled squares), andP95aR (filled circles) each titrated against fixed monolayers of theH3396 tumor cell line and detected with goat anti-human kappa alkalinephosphatase.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to tumor-specific human monoclonal antibodiesand functional fragments thereof. The human monoclonal antibodies andfunctional fragments thereof of the invention specifically bind toneoplastic cells compared to normal cells, and can be used forselectively targeting tumors. These antibodies are human in origin, andare unlikely to generate an immune response upon administration to ahuman subject. Therefore, they can be conjugated to cytotoxic orcytostatic agents and used to selectively target cancer cells for theelimination of tumors. The tumor-specific human monoclonal antibodiescan also be used in diagnostic procedures to identify neoplastic cells.Early detection of cancer greatly increases the chances of an individualsurviving the disease.

In one embodiment, the invention provides methods for generatinghybridomas producing tumor-specific human monoclonal antibodies. Normalhuman splenocytes are immunized in vitro with tumor cells or tumor cellmembranes in a mixed lymphocyte reaction. Such immunized splenocytes arethen immortalized to produce hybridomas providing an unlimited supply oftumor-specific human monoclonal antibodies. Using normal cells as thesource of lymphocytes greatly enlarges the repertoire of differenttumor-specific antibodies that can be obtained for the treatment ofcancer. Additionally, the type of cell or cell membrane used as theantigen in the method of the invention can be varied as needed toefficiently produce antibodies for different human therapeutic anddiagnostic applications.

In another embodiment, the invention is directed to nucleic acidsencoding human tumor-specific monoclonal antibodies. The nucleic acidscan be used to express the encoded human antibodies or fragmentsthereof. Additionally, the encoding nucleic acids can be recombinantlyengineered to produce modified human antibodies or functional fragmentswhich exhibit higher affinity or higher selectivity or tumor cells or toaugment other functional characteristics of the encoded antibodies. Suchmodified antibodies can additionally be constructed to contain othertherapeutically advantageous modifications, such as enhanced associationwith cytotoxic agents or increased stimulation of the immune system.

In a further embodiment, the invention is directed to antigensrecognized by the tumor-specific human monoclonal antibodies. Theantigens can be used for cancer diagnostic procedures and to developspecific S cytotoxic reagents for cancer therapy. Tumor-specificantigens can also be used as a vaccine and administered to individualsat risk of cancer to develop an effective immune response againstneoplastic cells. The nucleic acids encoding the tumor-specific antigenscan be used as probes in diagnostic procedures, or modified byrecombinant methods to develop specific inhibitors of the antigen.

The basic structure of an immunoglobulin or antibody molecule consistsof two identical light chains and two identical heavy chains, whichassociate non-covalently and can also be linked by disulfide bonds. Eachheavy and light chain contains an amino-terminal variable region ofabout 110 amino acids and constant sequences in the remainder of thechain. The variable region includes several hypervariable regions, orcomplementarity-determining regions (CDRs) that form the antigen-bindingsite of the antibody molecule and determine its specificity. On eitherside of the CDRs of the heavy and light chains is the framework region,a relatively conserved sequence of amino acids that anchors and orientsthe CDRs. The constant region consists of one of five heavy chainsequences (μ, γ, δ, α, or ε) and one of two light chain sequences (κorλ). The heavy chain constant region sequences determine the isotype ofthe antibody and the effector functions of the molecule.

As used herein, the term “human monoclonal antibody” is intended to meana monoclonal antibody comprising substantially the same heavy and lightchain CDR amino acid sequences as found in a particular humanimmunoglobulin. An amino acid sequence which is Em substantially thesame as a heavy or light chain CDR exhibits a considerable amount orextent of sequence identity when compared to a reference sequence andcontributes favorably to specific binding of an antigen boundspecifically by an antibody having the reference sequence. Such identityis definitively known or recognizable as representing the amino acidsequence of the particular human monoclonal antibody. Substantially thesame heavy and light chain CDR amino acid sequence can have, forexample, minor modifications or conservative substitutions of aminoacids so long as the ability to bind a particular antigen is maintained.The term “human monoclonal antibody” is intended to include a monoclonalantibody with substantially human CDR amino acid sequences produced, forexample, by recombinant methods, by lymphocytes or by hybridoma cells.

As used herein, the term “substantially the same” when used in referenceto an amino acid sequence is intended to mean an amino acid sequencethat has sufficient structural identity or similarity to a referenceamino acid sequence to be considered by one skilled in the art to have aspecific function of the polypeptide or fragment thereof encoded by thereference amino acid sequence. The term can include primary structuralidentity or similarity which is also referred to in the art as sequenceidentity or sequence similarity, respectively. An amino acid sequencethat is substantially the same as a reference sequence of an antibody,or functional fragment thereof can have a primary structure that is atleast 70% identical to a reference sequence including, for example,sequences that are at least 80%, at least 83%, at least 85%, at least90%, at least 95%, at least 97% or at least 98% identical to a referencesequence. The term can include tertiary structural identity orsimilarity, where tertiary structure is understood to refer to thethree-dimensional structure of a functionally active antibody orfragment thereof. A specific function included in the term can be anybiological activity that is specific to the polypeptide or fragmentthereof encoded by the reference amino acid sequence including, forexample, specific binding to a neoplastic cell or antigen thereof.

A nucleic acid sequence that is substantially the same as a referencesequence includes one that encodes the same polypeptide amino acidsequence. Nucleic acid sequences that are different from each other butencode identical amino acid sequences are commonly referred to in theart as having silent differences due to degeneracy of the genetic code.The term can include a full sequence or any portion thereof such as aparticular codon. Substantially identical amino acid or nucleic acidsequences can be identified using methods described below.

As used herein, the term “CDR” is intended to mean the non-contiguousantigen combining sites found within the variable region of both heavyand light chain polypeptides. This particular region has been describedby Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983) and by Chothia et al., J.Mol. Biol. 196:901-917 (1987) and additionally by MacCallum et al., J.Mol. Biol. 262:732-745 (1996), which are incorporated herein byreference, where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orfunctional fragment thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth,below in Table I as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe structure of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody. Those skilled in the art cancompare two or more antibody sequences by defining regions or individualamino acid positions of the respective sequences with the same CDRdefinition as described in further detail below. TABLE I CDR DefinitionsKabat¹ Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35 V_(H) CDR2 50-6552-56 47-58 V_(H) CDR3  95-102  95-102  93-101 V_(L) CDR1 24-34 24-3430-36 V_(L) CDR2 50-56 50-56 46-55 V_(L) CDR3 89-97 89-97 89-96¹Residue numbering follows the nomenclature of Kabat et al., supra²Residue numbering follows the nomenclature of Chothia et al., supra³Residue numbering follows the nomenclature of MacCallum et al., supra

As used herein, the term “functional fragment”, when used in referenceto a human monoclonal antibody, is intended to refer to a portion of themonoclonal antibody which is capable of specifically binding an antigenthat is specifically bound by the reference antibody. A functionalactivity can also be, for example, an effector function provided by anantibody constant region. Human monoclonal antibody functional fragmentsinclude, for example, individual heavy or light chains and fragmentsthereof, such as VL, VH and Fd; monovalent fragments, such as Pv, Fab,and Fab′; bivalent fragments such as F(ab′)₂; single chain Fv (scFv); Fcfragments and CDR domains. Such terms are described in, for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1989); Molec. Biology and Biotechnology: AComprehensive Desk Reference (Myers, R. A. (ed.), New York: VCHPublisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993);Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York,N.Y. (1990), which are incorporated herein by reference. The termfunctional fragment is intended to include, for example, fragmentsproduced by protease digestion or reduction of a human monoclonalantibody and by recombinant DNA methods known to those skilled in theart.

The term “VL fragment,” as used herein, refers to a fragment of thelight chain of a human monoclonal antibody which includes all or part ofthe light chain variable region, including the CDRs. A VL fragment canfurther include light chain constant region sequences.

The term “Fd fragment,” as used herein, refers to a fragment of theheavy chain of a human monoclonal antibody which includes all or part ofthe heavy chain variable region, including the CpRs. An Fd fragment canfurther include heavy chain constant region sequences.

The term “Fv fragment,” as used herein, refers to a monovalentantigen-binding fragment of a human monoclonal antibody, including allor part of the variable regions of the heavy and light chains, andabsent of the constant regions of the heavy and light chains. Thevariable regions of the heavy and light chains include, for example, theCDRs. For example, an Fv fragment includes all or part of the aminoterminal variable region of about 110 amino acids of both the heavy andlight chains.

The term “Fab fragment,” as used herein, refers to a monovalentantigen-binding fragment of a human monoclonal antibody that is largerthan an Fv fragment. For example, an Fab fragment includes the variableregions, and all or part of the first constant domain of the heavy andlight chains. Thus, a Fab fragment additionally includes, for example,amino acid residues from about 110 to about 220 of the heavy and lightchains.

The term “Fab′ fragment,” as used herein, refers to a monovalentantigen-binding fragment of a human monoclonal antibody that is largerthan a Fab fragment. For example, a Fab′ fragment includes all of thelight chain, all of the variable region of the heavy chain, and all orpart of the first and second constant domains of the heavy chain. Forexample, a Fab′ fragment can additionally include some or all of aminoacid residues 220 to 330 of the heavy chain.

The term “F(ab′)₂ fragment,” as used herein, refers to a bivalentantigen-binding fragment of a human monoclonal antibody. A F(ab′)₂fragment includes, for example, all or part of the variable regions oftwo heavy chains and two light chains, and can further include all orpart of the first constant domains of two heavy chains and two lightchains.

One skilled in the art knows that the exact boundaries of a fragment ofa human monoclonal antibody can be variable, so long as the fragmentmaintains a functional activity. Using well-known recombinant methods,one skilled in the art can engineer a nucleic acid to express afunctional fragment with any endpoints desired for a particularapplication.

As used herein, the term “label” is intended to mean a moiety that canbe attached to a human monoclonal antibody or other molecule of theinvention. Moieties can be used, for example, for therapeutic ordiagnostic procedures.

Therapeutic labels include, for example, moieties that can be attachedto a molecule of the invention and used to reduce the uncontrolledproliferation or viability of a neoplastic cell. A label which candecrease cell proliferation or viability can be, for example, acytotoxic or cytostatic agent, growth factor, cell death receptoragonist or immune modulator.

Diagnostic labels include, for example, moieties which can be detectedby analytical methods. Analytical methods include, for example,qualitative and quantitative procedures. Qualitative analytical methodsinclude, for example, immunohistochemistry and indirectimmunofluorescence. Quantitative analytical methods include, forexample, immunoaffinity procedures such as radioimmunoassay, ELISA orFACS analysis. Analytical methods also include both in vitro and in vivoimaging procedures. Specific examples of diagnostic labels that can bedetected by analytical means include enzymes, radioisotopes,chromophores, fluorochromes, chemiluminescent markers, and biotin.

A label can be attached directly to a molecule of the invention, or beattached to a secondary binding agent that specifically binds a moleculeof the invention. Such a secondary binding agent can be, for example, asecondary antibody. A secondary antibody can be either polyclonal ormonoclonal, and of human, rodent or chimeric origin.

As used herein, the term “cytotoxic or cytostatic agent” is intended tomean an agent which reduces the viability or proliferative potential ofa cell. Such agents can be attached, for example, to a human monoclonalantibody or other molecule of the invention and used to target cells ortissues. The targeted cells and tissues can include, for example,neoplastic cells and tumors. Examples of targeted cells and tissuesinclude those derived from breast, sung and ovarian tissue. Cytotoxic orcytostatic agents can function in a variety of ways to reduce cellviability or proliferation. Such functions include, for example,inhibiting DNA synthesis, inhibiting cell division, inducing apoptosis,or inducing non-apoptotic cell killing. Specific examples of cytotoxicand cytostatic agents include pokeweed antiviral protein, abrin, ricinand each of their A chains, doxorubicin, cisplastin, Iodine-131,Yttrium-90, Rhenium-188, Bismuth-212, Taxol, 5-Fluorouracil VP-16,Bleomycin, methotrexate, vindesine, adriamycin, vincristine,vinblastine, BCNU, mitomycin and cyclophosphamide and certain cytokinessuch as TNF-α and TNF-β. Thus, cytotoxic or cytostatic agents caninclude, for example, radionuclides, chemotherapeutic drugs, proteinsand lectins.

As used herein, the term “specific binding” is intended to mean aselective interaction of a human monoclonal antibody or functionalfragment thereof with an antigen. For such an interaction to beselective, a human monoclonal antibody will not substantially bind, orcan be made to not substantially bind, to markers other than theparticular antigen. Specific binding can include, for example,association constants (K_(a)) of about 10⁵ M⁻¹ or higher. Thus, specificbinding can include an association constant that is at least about 1×10⁶M⁻¹, 1×10⁷ M⁻¹, or 1×10⁸ M⁻¹. 1×10⁹ M⁻¹, 1×10¹⁰ M⁻¹, 1×10¹¹ M⁻¹ or1×10¹² M⁻¹. Specific binding can also include, for example, high avidityinteractions.

As used herein, the term “cancer” refers to a pathological conditioncharacterized by the presence of neoplastic cells. Neoplastic cells arecells that exhibit an abnormal morphological or proliferative phenotype.Such cells are characterized by, for example, anchorage independent cellgrowth, proliferation in reduced-serum medium, and loss of contactinhibition. Such cells are also characterized by, for example, abnormalnew growth of tissue, such as a tumor, angiogenic vasculature, andinvasion into surrounding tissue. Neoplastic cells can also metastasizefrom a primary tumor to other sites in the body. For example, a tumor ofthe breast, lung or ovary can metastasize to other organs yet still berecognizable as being comprised of breast, lung or ovarian cells. Thus,the term “tumor” or “cancer” in reference to breast, lung or ovary isintended to include metastases of these tumors to other organs of thebody.

As used herein, the term “effective amount” is intended to mean theamount of a molecule of the invention which can reduce proliferation ofneoplastic cells. The actual amount considered to be an effective amountfor a particular application can depend, for example, on such factors asthe affinity, avidity, stability, bioavailability or selectivity of themolecule, the moiety attached to the molecule, the pharmaceuticalcarrier and the route of administration. Effective amounts can bedetermined or extrapolated using methods known to those skilled in theart. Such methods include, for example, in vitro assays with culturedcells or tissue biopsies and credible animal models.

As used herein the term “isolated,” when used in the reference to anantibody, means separated from one or more compound that is found withthe antibody in nature or in a synthetic reaction used to produce theantibody including, for example, a reagent, precursor or other reactionproduct. An isolated agent can also include a substantially pure agent.The term can include naturally occurring molecules such as products ofbiosynthetic reactions or synthetic molecules.

The invention provides a human monoclonal antibody or functionalfragment having at least one CDR with substantially the amino acidsequence of a CDR of SEQ ID NO:2 or SEQ ID NO:4. The invention alsoprovides a human monoclonal antibody or functional fragment having atleast one CDR with substantially the amino acid sequence of a CDR of SEQID NO:6 or SEQ ID NO:8. The invention further provides human monoclonalantibodies or functional fragments thereof produced by the hybridomacell lines H1140, H2420 and H935. The hybridoma cell lines H1140, H2420and H935 are also provided.

The human monoclonal antibodies produced by the hybridoma cell linesLH11238, LH13, H1140, H2420 and H935 all exhibit specific binding toneoplastic cells as compared to normal cells and, therefore, aretumor-specific human monoclonal antibodies. In particular, the humanmonoclonal antibodies of the invention all selectively bind breastcarcinoma cells and show relatively little binding to normalfibroblasts. For example, the LH11238 antibody specifically binds to anantigen present on the surface and lysosomal compartments of breast andovarian carcinoma cells, as compared to normal fibroblasts, peripheralblood lymphocytes, melanoma cells or lung carcinoma cells. The LH13antibody specifically binds a product produced by breast, lung andovarian carcinoma cells, as compared to normal fibroblasts and melanomacells.

The human monoclonal antibodies produced by hybridoma lines H1140,H2420, H935 and LH13 are of the IgM isotype and A light chain class,whereas human monoclonal antibodies produced by hybridoma line LH11238is of the IgM isotype and κ light chain class. Further properties of thetumor-specific human monoclonal antibodies are described below in theExamples.

The nucleotide sequence encoding the heavy chain variable region (VH) ofthe human monoclonal antibody produced by LH11238 cell line has beendetermined and is designated SEQ ID NO:1. The VH amino acid sequence ofthe human monoclonal antibody produced by LH11238 cell line isdesignated SEQ ID NO:2. The nucleotide sequence encoding the light-chainvariable region (VL) of the human monoclonal antibody produced byLH11238 cell line has also been determined and is designated SEQ IDNO:3. The VL amino acid sequence of the human monoclonal antibodyproduced by LH11238 cell line is designated SEQ ID NO:4.

The nucleotide sequence encoding the heavy chain variable region (VH) ofthe human monoclonal antibody-produced by LH13 cell line has beendetermined and is designated SEQ ID NO:5. The VH amino acid sequence ofthe human monoclonal antibody produced by LH13 cell-line is designatedSEQ ID NO:6. The nucleotide sequence encoding the light chain variableregion (VL) of the human monoclonal antibody produced by LH13 cell linehas also been determined and is designated SEQ ID NO:7. The VL aminoacid sequence of the human monoclonal antibody produced by LH13 cellline is designated SEQ ID NO:8.

The invention further provides an isolated human monoclonal antibody orfunctional fragment thereof, having at least one CDR with substantiallythe amino acid sequence of a CDR of a sequence selected from the groupconsisting of SEQ ID NOs:10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42 and 44, wherein the human monoclonal antibody orfunctional fragment thereof specifically binds a neoplastic cell orantigen thereof.

Variants of the human monoclonal antibodies produced by the hybridomacell lines of the invention, including, for example, the LH13 andLH11238 cell lines, can exhibit specific binding to neoplastic cells ascompared to normal cells, and are, therefore, functional tumor-specifichuman monoclonal antibodies. Functional variants of the human monoclonalantibodies produced by the hybridoma cell lines LH13 and LH11238 andmethods for producing them are described in Example VIII and Table 5.

Functional variants of the human monoclonal antibody produced by theLH13 cell line that specifically bind to an antigen of a neoplastic cellare listed in Table 5 and include, for example, the antibody fragmentproduced by clone S97G, S97T or S97N each of which has an unmodified VLand a VH modified at position 101 of the sequence set forth in SEQ IDNO:6, the modification being at position 97 of HCDR3 according to thenumbering system a of Kabat et al. The antibody fragment produced byclone S97G has a glycine at position 97 of Kabat HCDR3 as set forth inthe VH amino acid sequence of SEQ ID NO:10 which is encoded by thenucleotide sequence set forth in SEQ ID NO:9. The antibody fragmentproduced by clone S97T has a threonine at position 97 of Kabat HCDR3 asset forth in the VH amino acid sequence of SEQ ID NO:12 and is encodedby the nucleotide sequence set forth in SEQ ID NO:11. The antibodyfragment produced by clone S97N has an HCDR3 with an asparagine atposition 97 of Kabat HCDR3 as set forth in the VH amino acid sequence ofSEQ ID NO:14 and is encoded by the nucleotide sequence set forth in SEQID NO:13.

Other functional variants of the human monoclonal antibody produced bythe LH13 cell line also include the antibody fragment produced by cloneR91Y or R91F each of which has an unmodified VH and a VL modified atposition 90 of the sequence set forth in SEQ ID NO:8, the modificationbeing at position 91 of LCDR3 using the numbering system of Kabat et al.The antibody fragment produced by clone R91Y has a tyrosine at position91 of Kabat LCDR3 as set forth in the VL amino acid sequence of SEQ IDNO:16 which is encoded by the nucleotide sequence set forth in SEQ IDNO:15. The antibody fragment produced by clone R91F has an LCDR3 with aphenylalanine at position 91 of Kabat LCDR3 as set forth in the VL aminoacid sequence of SEQ ID NO:18 which is encoded by the nucleotidesequence set forth in. SEQ ID NO:17.

A functional variant of the human monoclonal antibody produced by theLH13 cell line can be modified at position 98 of the VL sequence setforth in SEQ ID NO:8 while retaining an unmodified VH as exemplified bythe antibody fragments produced by clones V97Y and V97F, themodification being at position 97 of LCDR3 using the numbering system ofKabat et al. The antibody fragment produced by clone V97Y has a tyrosineat position 97 of Kabat LCDR3 as set forth in the VL amino acid sequenceof SEQ ID NO:20 which is encoded by the nucleotide sequence set forth inSEQ ID NO:19. The antibody fragment produced by clone V97F has an LCDR3with a tyrosine at position 97 of Kabat LCDR3 as set forth in the VLamino acid sequence of SEQ ID NO:22 which is encoded by the nucleotidesequence set forth in SEQ ID NO:21.

Combinatorial variants of the human monoclonal antibody produced by theLH13 cell line that specifically bind to an antigen of a neoplastic cellare also listed in Table 5 and include, for example, the antibodyfragment produced by clone 4D5, 4E2, 4H7, 4G11 or 3G4. The antibodyfragment produced by clone 4D5 is modified to have a threonine atposition 97 of Kabat HCDR3 and a tyrosine at position 91 of Kabat LCDR3compared to the human monoclonal antibody produced by the LH13 cellline. The amino acid sequence of the 4D5 VH is set forth in SEQ ID NO:12and is encoded by the nucleotide sequence set forth in SEQ ID NO:11 andthe VL amino acid sequence is set forth in SEQ ID NO:16 and encoded bythe nucleotide sequence set forth in SEQ ID NO:15.

The combinatorial variant produced by clone 4E2 is modified to have athreonine at position 97 of Kabat HCDR3, a tyrosine at position 91 ofKabat LCDR3, and a phenylalanine at position 97 of Kabat LCDR3 comparedto the human monoclonal antibody produced by the LH13 cell line. Theamino acid sequence of the 4E2 VH is set forth in SEQ ID NO:12 and isencoded by the nucleotide sequence set forth in SEQ ID NO:11 and the VLamino acid sequence is set forth in SEQ ID NO:24 and encoded by thenucleotide sequence set forth in SEQ ID NO:23.

The combinatorial variant produced by clone 4H7 is modified to have athreonine at position 97 of Kabat HCDR3; a phenylalanine at position 91of Kabat LCDR3, and a phenylalanine at position 97 of Kabat LCDR3compared to the human monoclonal antibody produced by the LH13 cellline. The amino acid sequence of the 4H7 VH is set forth in SEQ ID NO:12and is encoded by the nucleotide sequence set forth in SEQ ID NO:11 andthe VL amino acid sequence is set forth in SEQ ID NO:26 and encoded bythe nucleotide sequence set forth in SEQ ID NO:25.

The combinatorial variant produced by clone 4G11 is modified to have aphenylalanine at position 91 of Kabat LCDR3, and a phenylalanine atposition 97 of Kabat LCDR3 compared to the human monoclonal antibodyproduced by the LH13 cell line. The amino acid sequence of the 4G11 VHis set forth in SEQ ID NO:6 and is encoded by the nucleotide sequenceset forth in SEQ ID NO:5 and the VL amino acid sequence is set forth inSEQ ID NO:26 and encoded by the nucleotide sequence set forth in SEQ IDNO:25.

The combinatorial variant produced by clone 3G4 is modified to have atyrosine at position 91 of Kabat LCDR3, and a phenylalanine at position97 of Kabat LCDR3 compared to the human monoclonal antibody produced bythe LH13 cell line. The amino acid sequence of the 3G4 VH is set forthin SEQ ID NO:6 and is encoded by the nucleotide sequence set forth inSEQ ID NO:5 and the VL amino acid sequence is set forth in SEQ ID NO:24and encoded by-the nucleotide sequence set forth in SEQ ID NO:23.

Functional variants of the human monoclonal antibody produced by theLH11238 cell line that bind to a neoplastic cell are listed in Table 5and include, for example, the antibody fragment produced by clone Q89L,Q89G, Q89V, Q89F or Q89W each of which has an unmodified VH and a VLmodified at position 97 of the sequence set forth in SEQ ID NO:4, themodification being at position 89 of LCDR3 according to the numberingsystem of Kabat et al. The antibody fragment produced by clone Q89L hasa luecine at position 89 of Kabat LCDR3 as set forth in the VL aminoacid sequence of SEQ ID NO:28, which is encoded by the-nucleotidesequence set forth in SEQ ID NO:27. The antibody fragment produced byclone Q89G has an LCDR3 with a glycine at position 89 of Kabat LCDR3 asset forth in the VL amino acid sequence of SEQ ID NO:30, which isencoded by the nucleotide sequence set forth in SEQ ID NO:29. Theantibody fragment produced by clone Q89V has an LCDR3 with a valine atposition 89 of Kabat LCDR3 as set forth in the VL amino acid sequence ofSEQ ID NO:32, which is encoded by the nucleotide sequence set forth inSEQ ID NO:31. The antibody fragment produced by clone Q89F has an LCDR3with a phenylalanine at position 89 of Kabat LCDR3 as set forth in theVL amino acid sequence of SEQ ID NO:34, which is encoded by thenucleotide sequence set forth in SEQ ID NO:33. The antibody fragmentproduced by clone Q89W has a tryptophon at position 89 of Kabat LCDR3 asset forth in the VL amino acid sequence of SEQ ID NO:36, which isencoded by the nucleotide sequence set forth in SEQ ID NO:35.

Other functional variants of the human monoclonal antibody produced bythe LH11238 cell line also include the antibody fragment produced byclone P95aF or P95aR each of which has an unmodified VH and a VLmodified at position 103 of the sequence set forth in SEQ ID NO:4, themodification being at position 95 of LCDR3 according to the numberingsystem of Kabat et al. The antibody-fragment produced by clone P95aF hasa phenylalanine at position 95 of Kabat LCDR3 as set forth in the VLamino acid sequence of SEQ ID NO:42 which is encoded by the nucleotidesequence set forth in SEQ ID NO:41. The antibody fragment produced byclone P95aR has a phenylalanine at position 95 of Kabat LCDR3 as setforth in the VL amino acid sequence of SEQ ID NO:44 which is encoded bythe nucleotide sequence set forth in SEQ ID NO:43 or SEQ ID NO:45.

A functional variant of the human monoclonal antibody produced by theLH11238 cell line can be modified within LCDR3 at position 100 of the VLsequence set forth in SEQ ID NO:4 while retaining an unmodified VH asexemplified by the antibody fragment produced by clone N93C. Theantibody, fragment produced by clone N93C has a cysteine at position 93of Kabat LCDR3 as set forth in the VL amino acid sequence of SEQ IDNO:38 and encoded by the nucleotide sequence set forth in SEQ ID NO:37.As shown for the antibody fragment produced by clone N94C, a functionalvariant of the human monoclonal antibody produced by the LH11238 cellline can retain an unmodified VH and be modified to have a cysteine atposition 101 of the VL amino acid sequence set forth in SEQ ID NO:40(position 94 of Kabat LCDR3) and encoded by the nucleotide sequence setforth in SEQ ID NO:39.

The hybridomas producing the tumor-specific human monoclonal antibodiesof the invention were generated by in vitro immunization of human spleencell cultures with breast carcinoma cells. Briefly, a mixed lymphocytereaction (MLR) was established by co-culturing single cell suspensionsisolated from allogeneic human spleens. Tumor-reactive lymphocytes weresubsequently enriched by incubating MLR cultures either with monolayersor with enriched plasma membranes of breast carcinoma cells. In order toprovide a permanent source of human monoclonal antibodies of theinvention, immunized lymphocytes were immortalized either by fusion withthe K6H6/B5 heteromyeloma cell line or by transformation with EBVfollowed by fusion with K6H6/B5 cells. The particular source of antigenand the immortalization procedures used to generate each of thehybridoma cell lines of the invention are described more fully below inthe Examples.

The tumor-specific human monoclonal antibodies of the invention can alsobe generated by methods known to those skilled in the art. These methodsinclude, for example, in vivo and in vitro enrichment of tumor-reactivelymphocytes. For example, an individual with a breast, lung or ovariantumor can possess lymphocytes which express antibodies that specificallybind tumor-specific antigens, including, for example, LH13, LH11238,H1140, H2420 or H935 antigens. Such lymphocytes can be isolated, forexample, from the peripheral blood or from the spleen of the patient,and immortalized as described below.

Methods are also well known in the art for in vitro enrichment oftumor-reactive human lymphocytes, using tumor-specific antigens. Thesource of antigen can be, for example, substantially pure antigen, tumorcells or tumor cell fractions. A substantially pure antigen can beprepared by any of the methods well known to those skilled in the art,including, for example, chromatography, electrophoretic separation andimmuno-isolation.

An antigen useful for preparing human monoclonal antibodies of theinvention can be, for example, neoplastic cells. The neoplastic cellscan be, for example, cells obtained directly from tumors, culturedprimary tumor cells or established cell lines. Such cells can originatefrom any organ, tissue or fluid of the body, including, for example,breast, lung or ovary. The cancer cells can be untreated, fixed orgrowth-arrested. The fixation can be by any number of methods known tothose skilled in the art, including, for example, chemical fixation.Useful chemicals for fixation include, for example, paraformaldehyde,glutaraldehyde, methanol, or acetone. The cells can alternatively begrowth-arrested using cytostatic agents. A specific example of acytostatic agent is mitomycin C.

An antigen useful for preparing human monoclonal antibodies of theinvention can can also be a fraction of tumor cells. The tumor cellfraction can be, for example, cellular membranes, cytoplasmic contents,or nuclei. Methods for cell fractionation are well known in the art. Anantigen for preparing monoclonal antibodies of the invention can also bean antigen secreted by tumor cells. Such an antigen can be prepared, forexample, by isolation of conditioned medium or cell matrix of tumorcells using procedures known in the art.

An antigen prepared as described above can be used to stimulate humanlymphocytes to generate the tumor-specific human monoclonal antibodiesof the invention. Human lymphocytes can be obtained, for example, fromthe peripheral blood of live individuals, or from the spleen ofindividuals who are deceased or undergoing surgery. Lymphocytes can becultured with antigen directly. Alternatively, lymphocytes can becultured with antigen in a mixed lymphocyte reaction in the presence ofallogeneic lymphocytes. Appropriate culturing conditions for aparticular antigen and lymphocyte source can readily be determined bythose skilled in the art.

If desired, antigen-primed lymphocytes enriched by in vivo or in vitrostimulation as described above can be immortalized by any of a number ofprocedures known to those skilled in the art. Immortalization provides apermanent source of tumor-specific human monoclonal antibodies.Immortalization of lymphocytes can be accomplished by, for example,fusion with an immortal cell line. Such immortal cell lines useful forcell fusion can be, for example, human myeloma cells or humanlymphoblastoid B cell lines. Fusion partners can also be rodent myelomacells or human:rodent heteromyeloma cell lines. The heteromyeloma cellline can be, for example, the human:mouse heterohybridoma cell lineK6H6/B6. Alternatively, antigen-primed lymphocytes can be immortalizedby viral transformation, using, for example, viruses. A useful virus forimmortalization by viral transformation is EBV. Antigen-primedlymphocytes can also be immortalized by viral transformation followed byfusion. Viral transformation followed by fusion can be, for example, EBVtransformation followed by fusion with the K6H6/B5 cell line. Cultureconditions for lymphocyte fusion or viral transformation can readily bedetermined by those skilled in the art.

Immortalized lymphocytes can be screened for the production of humanmonoclonal antibodies that specifically bind to human tumor cells usingimmunoassays known to those skilled in the art. Such immunoassaysinclude, for example, quantitative and qualitative immunoassays.Qualitative immunoassays include, for example, precipitin methods,agglutinin reactions, immunohistochemistry, immunofluorescence,immunoblotting and immunoprecipitation. Quantitative immunoassaysinclude, for example, immunoaffinity methods such as radioimmunoassay,FACS analysis, and ELlSA analysis. ELISA analysis can be direct,sandwich or competitive. Immunoassays can be direct, using, for example,a labeled human monoclonal antibody. Such methods can alternatively beindirect, using, for example, a labeled anti-human secondary antibody.The label can be, for example, a fluorescent label, an enzyme, aradioisotope, or biotin. Detection can be by spectrophotometric,radiographic or chemiluminescent means, depending on the immunoassay.Such methods can also be used to screen for an improved antibody orfunctional fragment thereof having increased affinity for a neoplasticcell or antigen thereof.

An improved antibody or functional fragment thereof can also beidentified in a screen for association rate with a neoplastic cell orantigen thereof. Specifically, an improved antibody or functionalfragment thereof can be identified as one that has an increase inassociation rate compared to the parent antibody from which it isderived. Using such a screen an antibody or functional fragment thereofhaving improved therapeutic potency due to increased association ratecan be distinguished from a binding polypeptide that has an increasedfor the same antigen due to a decreased dissociation rate which is notcorrelated with therapeutic potency. In this regard, those skilled inthe art will recognize that according to the relationshipK_(A)=k_(on)/k_(off) an increased K_(A) can be due to increased k_(on)or decreased k_(off) or both.

An association rate can be determined in any non-equilibrium mixtureincluding, for example, one formed by rapidly contacting an antibody orfunctional fragment thereof with an antigen or by rapidly changingtemperature of a solution containing the binding partners. Anon-equilibrium mixture can be a pre-equilibrium mixture. Apre-equilibrium mixture can be formed, for example, by contacting asoluble antibody or functional fragment thereof and soluble antigen in acondition where the amount of total antigen and total antibody orfunctional fragment thereof in the detection chamber are constant.Measurements of association rates in pre-equilibrium mixtures can bemade in formats providing rapid mixing of antibody or functionalfragment thereof with antigen and rapid detection of changing propertiesof the antibody or functional fragment thereof or antigen on a timescaleof milliseconds or faster. Stopped flow and rapid quench flowinstruments such as those described below provide a convenient means tomeasure non-equilibrium kinetics. The association rate can also bemeasured in non-equilibrium mixtures including, for example, solutionscontaining insoluble species of antibody or functional fragment thereofor solutions containing variable concentrations of total antigen ortotal antibody or functional fragment thereof. Measurement of anassociation rate in a non-equilibrium mixture can be made in formatsproviding attachment of an antigen to a surface and continuous flow of asolution containing the antibody or functional fragment thereof over thesurface, or vice-versa, combined with,rapid detection of changingproperties of the antibody or functional fragment thereof, antigen orsurface such that measurements are made on a timescale of millisecondsor faster.

Formats for measuring association rates in pre-equilibrium mixturesinclude, for example, stopped flow kinetic instruments and rapid quenchflow instruments. A stopped flow instrument can be used to pushsolutions containing an antibody or functional fragment thereof andantigen from separate reservoirs into a mixing chamber just prior topassage into a detection cell. The instrument can then detect a changein one or more of the above described properties to monitor progress ofthe binding event. A rapid quench flow instrument can be used to rapidlymix a solution containing an antibody or functional fragment thereofwith a solution containing an antigen followed by quenching the bindingreaction after a finite amount of time. A change in one or more of theabove described properties can then be detected for quenched mixturesproduced by quenching at different times following mixing. Quenching canbe performed for example by freezing or addition of a chemical quenchingagent so long as the quenching step does not inhibit detection of theproperty relied upon for measurement of binding rate. Thus, a rapidquench instrument can be useful, for example, in situations wherespectroscopic detection is not convenient. A variety of instruments arecommercially available from vendors such as KinTek Corp. (State College,Pa.) and Hi-Tech Scientific (Salisbury, UK).

Formats for measuring association rates in non-equilibrium mixturesinclude, for example, surface plasmon resonance and evanescent waveinstruments. Surface plasmon resonance and evanescent wave technologyutilize an antigen or an antibody or functional fragment thereofattached to a biosensor surface. A solution containing the respectivebinding partner is passed over the biosensor surface and the change inrefractive index of the solution that occurs at the surface of a chipupon binding can be measured in a time dependent fashion. For example,surface plasmon resonance is based on the phenomenon which occurs whensurface plasmon waves are excited at a metal/liquid interface. Light isdirected at, and reflected from, the side of the surface not in contactwith sample, and SPR causes a reduction in the reflected light intensityat a specific combination of angle and wavelength. Biomolecular bindingevents cause changes in the refractive index at the surface layer, whichare detected as changes in the SPR signal. The binding event can beeither binding association or disassociation between a receptor-ligandpair. The changes in refractive index can be measured essentiallyinstantaneously and therefore allows for determination of the individualcomponents of an affinity constant. More specifically, the methodenables accurate measurements of an association rate constant (k_(on))and disassociation rate constant (k_(off)) Surface plasmon resonanceinstruments are available in the art including, for example, the BIAcoreinstrument, IBIS system, SPR-CELLIA system, Spreeta, and Plasmon SPR andevanescent wave technology is available in the Iasys system asdescribed, for example, in Rich and Myszka, Curr. Opin. Biotech.11:54-61 (2000).

The association rate can be determined by measuring a change in aproperty of a ligand or binding polypeptide at one or more discreet timeintervals during the binding event using, for example, the methodsdescribed above. Measurements determined at discreet time intervalsduring the binding event can be used to determine a quantitative measureof association rate or a relative measure of association rate.Quantitative measures of association rate can include, for example, anassociation rate value or k_(on) value. Quantitative values ofassociation rate or k_(on) can be determined from a mathematical orgraphical analysis of a time dependent measurement. Such analyses arewell known in the art and include algorithms for fitting data to a sumof exponential or linear terms or algorithms for computer simulation tofit data to a binding model as described for example in Johnson, Cur.Opin. Biotech: 9:87-89 (1998).

Association rates can be determined from mixtures containing insolublespecies or variable concentrations of total ligand or total bindingpolypeptide using mathematical and graphical analyses such as thosedescribed above if effects of mass transport are accounted for in thereaction. One skilled in the art can account for mass transport bycomparing association rates under conditions having similar limitationswith respect to mass transport or by adjusting the calculatedassociation rate according to models available in the art including, forexample those described in Myszka et al., Biophys. J. 75:583-594(19.98).

The tumor-specific antigen sample used for screening monoclonalantibodies for tumor reactivity need not be the same as the antigensample used to immunize the human lymphocytes. The antigen used inscreening can be, for example, substantially purified antigen, live orfixed tumor cell monolayers, live or fixed tumor cell suspensions,fractions of tumor cells, or sections of tumor biopsies, depending onthe assay procedure employed. The tumor cells can be, for example, humanbreast, ovarian or lung carcinoma cells.

The tumor-specific human monoclonal antibodies and functional fragmentsof the invention do not bind, bind only minimally, or can be made tobind only minimally to normal cell antigens. Normal cells or fractionsof normal cells can be used as controld for screening human monoclonalantibodies. Such normal cells can be, for example, live or fixed normaltissues or cultured cell lines. Cultured normal cell lines that can beused as controls include, for example, human fibroblasts and peripheralblood lymphocytes. Although any normal cell can be used as a control,the selection of a particular control will be based, in part, on thespecificity of the particular tumor-specific monoclonal antibody whichis being screened. For example, if a human tumor-specific monoclonalantibody is produced and screened against a. carcinoma cell, then onetype of normal cell which can be used as a comparison is a normalepithelial cell culture or cell line. Similarly, if a humantumor-specific monoclonal antibody is produced and screened against asarcoma cell, then one type of normal cell which can be used as acomparison is a normal fibroblast cell culture or cell line. A normalcell culture or cell line from the same tissue type or from the sameindividual can also provide a normal control. Those skilled in the artwill know what is an appropriate type of normal cell to be used as acontrol to determine specific binding to a particular type of tumorcell.

Tumor-specific human monoclonal antibodies or functional fragmentsthereof can be purified and quantitated for use in immunodiagnostic andimmunotherapeutic procedures. Such purification methods are well knownto those skilled in the art and depend on the source of human monoclonalantibodies and the particular application. Purification methods caninclude, for example, precipitation, electophoresis, chromatography, andimmunoaffinity purification. The purified antibodies can be quantitatedin comparison with known standard controls, using, for example,spectrophotometry or immunoassays known in the art.

To further characterize tumor-specific human monoclonal antibodies,their class and subclass can also be determined by immunoassays thatmeasure the presence of individual heavy and light chain polypeptides.Such immunoassays include ELISA assays and are known to those skilled inthe art.

The invention further provides functional fragments of thetumor-specific human monoclonal antibodies of the invention. Afunctional fragment of a human monoclonal antibody maintains abiological activity, such as specific binding or an effector function. Afunctional fragment can therefore be beneficially used for the detectionand treatment of cancer.

Functional fragments include fragments with substantially the same heavyand light chain variable regions as a tumor-specific human monoclonalantibody of the invention. For example, functional fragments includefragments wherein at least one of the CDR sequences consist ofsubstantially the same amino acid sequence as the CDR sequences of atumor-specific human monoclonal antibody of the invention. Suchfunctional fragments include, for example, VL, Fd, Fv, Fab, Fab′,F(ab′)₂, Fc and CDR fragments. For example, a functional fragment couldhave one or more of the three CDR sequences of the VL, or one or more ofthe three CDR sequences of the VH, or a combination of VL and VH CDRs ofa human monoclonal antibody of the invention. The appropriate number andcombination of VH and VL CDR sequences can be determined by thoseskilled in the art depending on the desired affinity and specificity andthe intended use of the functional fragment.

Functional fragments of human monoclonal antibodies of the invention canreadily be produced and isolated using methods well known to thoseskilled in the art. Such methods include, for example, proteolyticmethods, recombinant methods and chemical synthesis. Proteolytic methodsfor the isolation of functional fragments use human monoclonalantibodies as starting material. Enzymes suitable for proteolysis ofhuman monoclonal antibodies include, for example, papain, pepsin andelastin. The appropriate enzyme can be readily chosen by one skilled inthe art, depending on, for example, whether monovalent or bivalentfragments are required. For example, papain cleavage results in twomonovalent Fab, fragments that bind antigen and an Fc. fragment. Pepsincleavage, for example, results in a bivalent F(ab′)₂ fragment. A F(ab′),fragment of the invention can further be reduced using, for example, DTTor β-mercaptoethanol to produce two monovalent Fab′ fragments.

Functional fragments produced by proteolysis can be purified by affinityand column chromatographic procedures. For example, undigestedantibodies and Fc fragments can often be removed by-binding to proteinA. Additionally, functional fragments can be purified by virtue of theircharge and size, using, for example, ion-exchange and gel filtrationchromatography. Such methods are well known to those skilled in the art.

Recombinant methods for producing functional fragments of humanmonoclonal antibodies begin with the isolated nucleic acid of desiredregions of the immunoglobulin heavy and light chains. Such regions caninclude, for example, all or part of the variable region of the heavyand light chains. Such regions can particularly include the CDRs of theheavy and light chains.

The invention provides an isolated nucleic acid encoding a humanmonoclonal antibody or functional fragment thereof, having a nucleotidesequence encoding substantially the amino acid sequence of at least oneCDR encoded by a nucleotide sequence selected from the group consistingof SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43 and 45. The invention further provides anisolated nucleic acid,encoding a variable region domain, having anucleotide sequence encoding substantially the amino acid sequence ofthe CDRE encoded by a nucleotide sequence selected from the groupconsisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43 and 45. Also provided is an isolatednucleic acid encoding a CDR, having substantially the amino acidsequence of a CDR encoding by SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45.

A nucleic acid encoding a human monoclonal antibody or functionalfragment of the invention can be produced using methods known to thoseskilled in the art. One useful procedure for isolating such DNA beginswith cDNA which can be reverse-transcribed from RNA of hybridoma cellsthat produce a tumor-specific human monoclonal antibody. Methods forcDNA synthesis are well known in the art. A cDNA encoding a functionalfragment of a heavy or light chain can be amplified using, for example,the polymerase chain reaction (PCR). The PCR technology is the subjectmatter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,292,all of which are incorporated by reference herein. Suitable primers forPCR are known and can be determined by those of skill in the art usingconserved sequences which flank the particular functional fragment of aheavy or light chain. Suitable PCR conditions are known and can alsosimilarly be determined by those skilled in the art.

A nucleic acid encoding a functional fragment of a human monoclonalantibody of the invention can also be directly synthesized by methods ofoligonucleotide synthesis known in the art. Alternatively, smallerfragments can be synthesized and joined to form a larger functionalfragment using recombinant methods known in the art.

Nucleic acids encoding a functional fragment of a human monoclonalantibody of the invention can be cloned into a suitable expressionvector and expressed in a suitable host. A suitable vector and host cellsystem can allow, for example, co-expression and assembly of functionalfragments of the heavy and light chains. Suitable systems for theexpression of antibody fragments can be determined by those skilled inthe art and include, for example, M13 phage immunoexpression vectors.Recombinant functional fragments of the invention can be substantiallypurified using methods known in the art, and which depend on theparticular vector and host expression system used.

Isolated nucleic acids encoding tumor-specific human monoclonalantibodies or functional fragments can also be engineered to produceantibodies with optimal properties such as affinity, selectivity,avidity, stability or bioavailability. Such modifications can include,for example, addition, deletion, or substitution of amino acid residuesor substitution of a D-amino acid or amino acid mimetic, so long as thefragment maintains a functional activity such as, for example, antigenbinding specificity.

In addition, the invention provides distinct libraries of LH13 andLH11238 variants. An LH13 library can contain variants having at leastone amino acid alteration in a Kabat light chain CDR3 (LCDR3)corresponding to positions 88-98 of SEQ ID NO:8, variants having atleast one amino acid alteration in a Kabat heavy chain CDR3 (HCDR3)corresponding to positions 99-107 of SEQ 1D NO:t. An LH111238 librarycan contain variants having at least one amino acid alteration in aKabat LCDR3 corresponding to positions 97-106 of SEQ ID NO:4. Althoughthe libraries exemplified above are based on the numbering system ofKabat et al., those skilled in the art will be able to produce similarlibraries based on any of the CDR definition including for example thoseof Chothia et al. or MacCallum et al using the numbering systems setforth previously. A library of the invention can contain,variants havingat least one amino acid alteration in a region corresponding one or moreof the CDRs of LH13 or LH11238 including HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 or LCDR3 according to the numbering systems set forth previously.A library of LH13 or LH11238 variants can be used to screen forantibodies or functional fragments having increased antigen orneoplastic cell binding activity as set forth in further detail below.

A library that is sufficiently diverse to contain an antibody variant orfragment thereof with enhanced binding affinity can be prepared by avariety of methods well known in the art. Those skilled in the art willknow what size and diversity of the library is necessary or sufficientfor the intended purpose. For example, a library of antibody or antibodyfragment variants can be prepared that contains each of the 19 essentialamino acids not found in the parent antibody at each of the positions inone or more CDR and the resultant population screened for variants withenhanced binding activity.

Alternatively, a focused library can be prepared utilizing thestructural, biochemical and modeling information relating to antibodiesas described herein. It is understood that any information relevant tothe determination or prediction of residues or regions important for thebinding activity or structural integrity or stability of an antibody ofthe invention can be useful in the design of a focused library. Thus,the antibody variants that make up the library can contain amino acidalterations at one or more amino acid positions located in a region orat a particular position determined or predicted to be important forbinding activity or structural integrity or stability of an antibodythat binds a neoplastic cell or antigen thereof. A focused library ofantibody variants provides the advantage of decreasing the number ofvariants that need to be screened in order to identify a variant withenhanced binding activity or structural stability.

Combinatorial approaches to synthesizing and screening focused librariescan further provide advantages in the speed and efficiency ofidentifying improved antibody variants or fragments thereof. Asdemonstrated in Example VIII, multiple amino acid changes in the LH13variants were additive. The additivity of amino acid changes in otherunrelated, mono,clonal antibodies has been reported previously asdescribed, for example, in Yelton et al., J. Immunol. 155:1994-2004(1995), Wu et al., supra (1998), and Wu et al., supra (1999). Theidentification of additive amino acid combinations by synthesis of acombinatorial library based on single amino acid changes discovered fromHCDR3 and LCDR3 libraries permitted rapid enhancement of the antibodyaffinity in a very efficient manner.

As demonstrated in Example VIII, the two step affinity maturation ofLH13 was accomplished with the synthesis of only 416 distinct proteinvariants consisting of 171 HCDR3 mutants and 209 LCDR3 mutants in afirst step followed by 36 combinatorial variants in step two. Incontrast, total randomization of the three CDR residues demonstrated toinfluence activity, LCDR3 S97, HCDR3 R91, and HCDR3 V97, would-haverequired the expression of a library containing 19³, or 6,659, variants.Thus, the combinatorial approach described in Example VIII providedstepwise improvement in affinity that captured additivity of independentmutations is an efficient method of simplifying the affinity maturationprocess. Those skilled in the art will recognize from the results ofExample VIII that the affinity of an antibody of the invention such asan enhanced class-switched variant can be further improved through thesynthesis of additional LH13 libraries corresponding to HCDR1 and HCDR2and LCDR1 and LCDR2. Moreover, the beneficial mutations identified fromthese libraries can be used in a combinatorial fashion to identifyimproved antibody or antibody fragment variants having increasedaffinity for a neoplastic cell or antigen thereof.

Individual residues or regions important for binding of an antibody ofthe invention to a neoplastic cell or antigen thereof can be determinedor predicted through a variety of methods known in the art and can beused to focus the synthesis of a library of antibody or antibodyfragment variants. Structural comparison of sequences (primarystructures) or three dimensional structures (tertiary structures) or acombination of both can be made between antibodies that bind the sameantigen or similar antigens to identify individual residues or regionsfor mutation. For example, structural modeling based on antibodytertiary structure can reveal the topological, electrostatic,hydrophobic or hydrophilic environment of a binding site or particularCDR involved in antigen binding. Comparison of shared properties ofbinding sites for two or more antibodies that bind a common antigen orstructurally similar antigen can be used to identify positions orregions for mutagenesis. Sequence comparison methods can be used toalign sequences and identify positions in two or more antibodies havingresidues that are homologous or share similar steric, electrostatic,hydrophobic or hydrophilic properties. A combination of primary andtertiary structure comparisons can be used to identify positions to bealtered. For example, positions located apart from each other in primarystructure which are important for binding can be identified in atertiary structure analysis and then emphasized in a primary sequencefor use in querying a database of antibody primary sequences orcomparing to one or more other antibody primary sequence.

Methods for comparing primary sequence structure which can be used todetermine that two sequences are substantially the same are well knownin the art. For example, one method for determining if two sequences aresubstantially the same is BLAST, Basic Local Alignment Search Tool,which can be used according to default parameters as described byTatiana et al., FEMS Microbial Lett. 174:247-250 (1999) or on theNational Center for Biotechnology Information web page atncbl.nlm.gov/BLAST/. BLAST is a set of similarity search programsdesigned to examine all available sequence databases and can function tosearch for similarities in amino acid or nucleic acid sequences. A BLASTsearch provides search scores that have a well-defined statisticalinterpretation. Furthermore, BLAST uses a heuristic algorithm that seekslocal alignments and is therefore able to detect relationships amongsequences which share only isolated regions of similarity including, forexample, protein domains (Altschul et al., J. Mol. Biol. 215:403-410(1990)).

In addition to the originally described BLAST (Altschul et al., supra,1990), modifications to the algorithm have been made (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). One modification is GappedBLAST, which allows gaps, either insertions or deletions, to beintroduced into alignments. Allowing gaps in alignments tends to reflectbiologic relationships more closely. For example, gapped BLAST can beused to identify sequence identity within similar domains of two or morepolypeptides. A second modification is PSI-BLAST, which is a sensitiveway to search for sequence homologs. PSI-BLAST performs an initialGapped BLAST search and uses information from any significant alignmentsto construct a position-specific score matrix, which replaces the querysequence for the next round of database searching. A PSI-BLAST search isoften more sensitive to weak but biologically relevant sequencesimilarities.

A second resource that can be used to determine if two sequences aresubstantially the same is PROSITE, available on the world wide web atExPASy. PROSITE is a method of determining the function ofuncharacterized polypeptides translated from genomic or cDNA sequences(Bairoch et al., Nucleic Acids Res. 25:217-221 (1997)). PROSITE consistsof a database of biologically significant sites and patterns that can beused to identify which known family of polypeptides, if any, the newsequence belongs. Using this or a similar algorithm, a polypeptide thatis substantially the same as another polypeptide can be identified bythe occurrence in its sequence of a particular cluster of amino acidresidues, which can be called a pattern, motif, signature orfingerprint, that is substantially the same as a particular cluster ofamino acid residues in a reference polypeptide including, for example,those found in similar domains. PROSITE uses a computer algorithm tosearch for motifs that identify polypeptides as family members. PROSITEalso maintains a compilation of previously identified motifs, which canbe used to determine if a newly identified polypeptide is a member of aknown family.

Sequence comparison can include a full sequence of a gene, cDNA orexpressed products thereof or can include one or more particular regionsthereof. A particular region can be identified by visual inspection of asequence alignment to identify regions of relatively high homology orsimilarity. Those regions can be cross referenced with structural datato find correlations between a particular structural domain and regionof homology, as described above. A structural model of a referenceantibody or fragment thereof can also be used in an algorithm thatcompares polypeptide structure including, for example, SCOP, CATH, orFSSP which are reviewed in Hadley and Jones, Structure 7:1099-1112(1999) and regions having a particular fold or conformation used as aregion for sequence comparison to a second polypeptide to identifysubstantially similar regions.

In addition to structural modeling of antibodies, biochemical data canbe used to determine or predict positions or regions of an antibody thatare important for binding a neoplastic cell or antigen thereof and thesepositions or regions targeted in preparing a focused library of antibodyvariants. In this regard, the characterization of a parent antibody orvariants thereof with respect to binding rate constant (k_(on)),dissociation rate constant (k_(off)) or equilibrium binding affinityconstant (k_(d) or k_(a)) is useful for identifying regions importantfor binding to a neoplastic cell or antigen thereof.

To generate a library of antibody variants distinct types of informationcan be used alone or combined to determine or predict a region of anamino acid sequence that is important for binding to a neoplastic cellor antigen thereof. For example, information based on structuralmodeling and biochemical analysis, such as comparison of affinities forvariants having changes at a structurally conserved position, can becombined to determine a region of an amino acid sequence of an antibodyimportant for binding activity. Because information obtained by avariety of methods can be combined to predict regions important forbinding, one skilled in the art will appreciate that the regionsthemselves represent approximations rather than strick confines. As aresult, a library of antibody variants can be altered at positionsoutside of the regions determined or predicted to directly interact withan antigen. For example, framework regions can be important forstructural stability of an antibody or fragment thereof or can influencethe binding affinity by long range or through space interactions thatinfluence binding site properties. Similarly, a variant of an antibodyor fragment thereof that binds a neoplastic cell or antigen thereof canhave amino acid alterations outside of the CDRs or other regionsidentified herein as directly involved in binding.

It is further understood that the number or location of amino acidpositions predicted to be important for binding activity can vary basedon the predictive methods used and the structures compared. For example,as described above, different CDR definitions can be used to compareantibody sequences. Those skilled in the art will understand that thesame CDR definition can be used to identify a region or position to becompared between two or more antibody structures. For example, two ormore antibodies can be compared with respect to the Kabat et al.definition of CDR3 as described in Example VIII and shown in Table V.Those skilled in the art can determine an appropriate CDR definition foruse in evaluating an antibody based on the primary or tertiary structureof the antibody. Similarly, a CDR definition to be used for comparingtwo or more antibody structures can be determined based on initialinspection of each antibody structure and identification of thedefinition which best fits all structures to be compared. Other factorscan also be considered in choosing an appropriate CDR definitionincluding, for example, functional properties of a parent antibody orvariants thereof. Those skilled in the art can perform separatecomparisons of the same antibodies using different CDR definitions andthereby identify regions or positions to be altered according to thefrequency with which the regions or positions are found to be similar orhomologous in the separate comparisons.

Methods for preparing libraries containing diverse populations ofvarious types of molecules such as peptides, peptoids andpeptidomimetics are well known in the art (see, for example, Ecker andCrooke, Biotechnology 13:351-360 (1995), and Blondelle et al., TrendsAnal. Chem. 14:83-92 (1995), and the references cited therein; see,also, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger'sMedicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; JohnWiley & Sons 1995), pages 803-861, and Gordon et al., J. Med. Chem.37:1385-1401 (1994)). Where a molecule is a peptide, protein or fragmentthereof, the molecule can be produced in vitro directly or can beexpressed from a nucleic acid, which can be produced in vitro. Methodsof synthetic peptide chemistry are well known in the art.

A library of antibody variants can be produced, for example, byconstructing a nucleic acid expression library encoding antibodyvariants. Methods for producing such libraries are well known in the art(see, for example, Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989);Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., ColdSpring Harbor Press, Plainview, N.Y. (2001); Ausubel et al. (CurrentProtocols in Molecular Biology (Supplement 47), John Wiley & Sons, NewYork (1999)). A library of nucleic acids encoding antibody variants canbe composed of DNA, RNA or analogs thereof. A library containing RNAmolecules can be constructed, for example, by synthesizing the RNAmolecules chemically.

The generation of a library of nucleic acids encoding antibody orantibody fragment variants can be by any means desired by the user.Those skilled in the art will know what methods can be used to generatelibraries of nucleic acids encoding antibody or antibody fragmentvariants. For example, libraries of antibody variants can be generatedby mutagenesis of a nucleic acid encoding a parent antibody such as LH13or LH11238 using methods well known to those skilled in the art(Sambrook et al., supra (1989); Sambrook et al., supra (2001); Ausubelet al., supra (1999)). A library of nucleic acids encoding antibody orantibody fragment variants of the invention can be randomized to besufficiently diverse to contain nucleic acids encoding every possiblenaturally occurring amino acid at each amino acid position of one ormore CDR. Alternatively, a library of nucleic acids can be prepared suchthat it contains nucleic acids encoding every possible naturallyoccurring amino acid at each amino acid only at positions located withina region of a CDR predicted or determined to be important for binding toa neoplastic cell or antigen thereof, as described in Example VIII.

One or more mutations can be introduced into a nucleic acid moleculeencoding an antibody or antibody fragment variant to yield a modifiednucleic acid molecule using, for example, site-directed mutagenesis (seeWu (Ed.), Meth. In Enzymol. Vol. 217, San Diego: Academic Press (1993);Higuchi, “Recombinant PCR” in Innis et al. (Ed.), PCR Protocols, SanDiego: Academic Press, Inc: (1990), each of which is incorporated hereinby reference): Such mutagenesis can be used to introduce a specific,desired amino acid alteration. Thus, distinct libraries containing aminoacid alterations in one or more of the regions determined to beimportant for binding lactivity as well as a single library containingmutations in several or all of the regions can be prepared.

The efficient synthesis and expression of libraries of antibody orantibody fragment variants using oligonucleotide-directed mutagenesiscan be accomplished as previously described by Wu et al., Proc. Natl.Acad. Sci. USA, 95:6037-6042 (1998); Wu et al., J. Mol. Biol.,294:151-162 (1999); and Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492(1985). Oligonucleotide-directed mutagenesis is a well-known andefficient procedure for systematically introducing mutations,independent of their phenotype and is, therefore, ideally suited fordirected evolution approaches to protein engineering. To performoligonucleotide-directed mutagenesis a library of nucleic acids encodingthe desired mutations is hybridized to single-stranded uracil-containingtemplate of the wild-type sequence. The methodology is flexible,permitting precise mutations to be introduced without the use ofrestriction enzymes, and is relatively inexpensive if oligonucleotidesare synthesized using codon-based mutagenesis.

Amino acid substitutions can be introduced by mutating nucleic acidcodons encoding the particular amino acid using methods known in theart. Single or multiple codons can be varied, so long as the fragmentretains a functional activity. Rapid methods for making and screeningmultiple simultaneous changes are well known within the art and can beused to produce a library of encoding nucleic acids which contain allpossible or all desired changes and then expressing and screening thelibrary for human monoclonal antibodies or fragments which retainfunction. Such methods include, for example, codon based mutagenesis,synthesis of stochastic oligonucleotides and partially degenerateoligonucleotide synthesis.

Codon based mutagenesis is the subject matter of U.S. Pat. Nos.5,264,563 and 5,523,388 and is advantageous for the above proceduressince it allows for the production of essentially any and all desiredfrequencies of encoded amino acid residues at any and all particularcodon positions within an oligonucleotide. Such desired frequenciesinclude, for example, the truly random incorporation of all twenty aminoacids or a specified subset thereof as well as the incorporation of apredetermined bias of one or more particular amino acids so as toincorporate a higher or lower frequency of the biased residues comparedto other incorporated amino acid residues.

Synthesis of stochastic oligonucleotides and partially degenerateoligonucleotide synthesis can similarly be used for producing andscreening for functionally equivalent amino acid changes. However, dueto the degeneracy of the genetic code, such methods can incorporateredundancies at a desired amino acid position (see, for example, U.S.Pat. No. 5,723,323). Stochastic oligonucleotide synthesis includes thecoupling of all four nucleotides at each nucleotide position within acodon. Other stochastic methods of synthesis also exist which can resultin degenerate or partially degenerate oligonucleotides oroligonucleotides which encode completely random amino acid sequences(see, for example, U.S. Pat. No. 5,723,323).

Partially degenerate oligonucleotide synthesis is the coupling of equalportions of all four nucleotides at the first two nucleotide positions,for example, and equal portions of two nucleotides at the thirdposition. Both of these latter synthesis methods can be found describedin, for example, Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382,(1990) and Devlin et al., Science 249:404-406, (1990).

A modification to the above-described codon-based synthesis forproducing a diverse number of variant sequences can similarly beemployed for the production of the libraries of antibody or antibodyfragment variants described herein. This modification is based on thetwo vessel method described above which biases synthesis toward theparent sequence and allows the user to separate the variants intopopulations containing a specified number of codon positions that haverandom codon changes.

Briefly, this synthesis is performed by continuing to divide thereaction vessels after the synthesis of each codon position into two newvessels. After the division, the reaction products from each consecutivepair of reaction vessels, starting with the second vessel, is mixed.This mixing brings together the reaction products having the same numberof codon positions with random changes. Synthesis proceeds by thendividing the products of the first and last vessel and the newly mixedproducts from each consecutive pair of reaction vessels and redividinginto two new vessels. In one of the new vessels, the parent codon issynthesized and in the second vessel, the random codon is synthesized.For example, synthesis at the first codon position entails synthesis ofthe parent codon in one reaction vessel and synthesis of a random codonin the second reaction vessel. For synthesis at the second codonposition, each of the first two reaction vessels is divided into twovessels yielding two pairs of vessels. For each pair, a parent codon issynthesized in one of the vessels and a random codon is synthesized inthe second vessel. When arranged linearly, the reaction products in thesecond and third vessels are mixed to bring together those productshaving random codon sequences at single codon positions. This mixingalso reduces the product populations to three, which are the startingpopulations for the next round of synthesis. Similarly, for the third,fourth and each remaining position, each reaction product population forthe preceding position are divided and a parent and random codonsynthesized.

Following the above modification of codon-based synthesis, populationscontaining random codon changes at one, two, three and four positions aswell as others can be conveniently separated out and used based on theneed of the individual. Moreover, this synthesis scheme also allowsenrichment of the populations for the randomized sequences over theparent sequence since the vessel containing only the parent sequencesynthesis is similarly separated out from the random codon synthesis.This method can be used to synthesize a library of nucleic acidsencoding antibody variants having amino acid alterations in one or moreCDR predicted to be important for binding to a neoplastic cell orantigen thereof.

Alternatively, a library of nucleic acids encoding antibody or antibodyfragment variants can also be generated using gene shuffling. Geneshuffling or DNA shuffling is a method for directed evolution that:generates diversity by recombination (see, for example, Stemmer, Proc.Natl. Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); Crameri et al., Nature 391:288-291 (1998); Stemmer et al., U.S.Pat. No. 5,830,721, issued Nov. 3, 1998). Gene shuffling or DNAshuffling is a method using in vitro homologous recombination of poolsof selected mutant genes. For example, a pool of point mutants of aparticular gene can be used. The genes are randomly fragmented, forexample, using DNase, and reassembled by PCR. If desired, DNA shufflingcan be carried out using homologous genes from different organisms togenerate diversity (Crameri et al., supra, 1998). The fragmentation andreassembly can be carried out in multiple rounds, if desired. Theresulting reassembled genes constitute a library of antibody or antibodyfragment variants that can be used in the invention compositions andmethods.

For certain therapeutic and diagnostic applications it may be preferableto use antibodies or fragments thereof with the same antigen specificitybut with different isotypic or allotypic determinants-. Such antibodiescould have, for example, decreased immunogenicity, increased stability,or more optimal effector functions. Thus, functional fragments of theinvention can include those obtained by cloning the CDR sequences of ahuman monoclonal antibody of the invention into different frameworkregions. Such different framework regions can be obtained from differentspecies, different human individuals, or different heavy or light chainclasses from the same or different individual. Such CDR grafting methodsare well known to those skilled in the art. An example of grafting CDRsequences from an IgM immunoglobulin to an IgG framework region isdescribed in Example VIII. As demonstrated in Example VIII, in vitroaffinity maturation can permit the improvement of virtually anyantibody, including low affinity IgMs with essentially germlinesequence.

Functional activity of antibody or antibody fragment variants of theinvention can be evaluated by, for example, methods described above fordetermining the immunoreactivity of human monoclonal antibodies.Particularly useful methods for determining functional activity offragments include competitive radioimmunoassay and competitive ELISAassay. Such methods can be used to screen for variants having improvedaffinity for a neoplastic cell or antigen thereof. The methods can beused at different stringency depending upon the number of variantsscreened. Stringency can be also adjusted based on the number of cyclesor iterations through which the screen has progressed. For example, lowstringency can be used in early screening steps and the stringencyincreased in later screening steps. Use of low stringent affinitycriteria in screening antibody libraries can be used to broaden thediversity being explored without creating larger libraries or screeninga greater number of clones and may lead to the discovery of novelantibodies from previously screened Ig repertoires.

The present invention provides pharmaceutical compositions containing ahuman monoclonal antibody or functional fragment of the invention and apharmaceutical carrier. Such compositions can be used to administer ahuman monoclonal antibody or fragment to reduce the proliferation orviability of neoplastic cells. Such compositions can also be used todetect neoplastic cells.

Suitable pharmaceutical carriers for the methods of the invention arewell known and include, for example, aqueous solutions such asphysiologically buffered saline, and other solvents or vehicles such asglycols, glycerol, oils or injectable organic esters. A pharmaceuticalcarrier can contain a physiologically acceptable compound that acts, forexample, to stabilize or increase the solubility of a pharmaceuticalcomposition. Such a physiologically acceptable compound can be, forexample, a carbohydrate, such as glucose, sucrose or dextrans; anantioxidant, such as ascorbic acid or glutathione; a chelating agent; alow molecular weight protein; or another stabilizer or excipient.Pharmaceutical carriers, including stabilizers and preservatives, aredescribed., for example, in Martin, Remington's Pharm. Sci., 15th Ed.(Mack Publ. Co., Easton, 1975), which is incorporated herein byreference. All of the above-described pharmaceutical carriers and mediacan be what is termed in the art pharmaceutical grade which means thatthey are of sufficient purity and quality for use in humans and aredistinguishable from comparable reagents in research grade formulations.

Those skilled in the art will know that the choice of the pharmaceuticalmedium and the appropriate preparation of the composition will depend onthe intended use and mode of administration.

The invention provides a variable heavy chain domain or functionalfragment thereof having CDRs with substantially the CDR sequences of theCDRS of a sequence selected from the group consisting of SEQ ID NOs:2,6, 10, 12, and 14. The invention further provides a variable light chaindomain or functional fragment thereof having the CDRs with substantiallythe CDR sequences of the CDRs of a sequence selected from the groupconsisting of SEQ ID NOs:4, 8, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42 and 44. The invention also provides a CDR or functionalfragment thereof having substantially the amino acid sequence of a CDRselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44.

A CDR or functional fragment thereof of the invention can be produced bymethods known in the art and as described above. For example, a CDR ofthe invention can be produced by recombinant means or chemicalsynthesis. A CDR of the invention can be advantageously used, forexample, to generate anti-idiotypic antibodies that selectively bind thetumor-specific human monoclonal antibodies of the invention. Methods forproducing and using anti-idiotypic antibodies for diagnostic andtherapeutic purposes are well known in the art.

An antibody fragment of the invention can be a variable heavy chaindomain or variable light chain domain. The fragment can be expressed ina recombinant system using methods described herein or a variety ofother methods known in the art. For example, the expression of variableheavy chain domains having affinities for a protein antigens similar tothose expected of monoclonal antibodies for protein, antigens are knownin the art as described, for example, in Ward et al., Nature 341:544-546(1989) and Williams et al., Proc Natl. Acad. Sci. USA 86:5537-5541(1989). A fragment of the invention can be modified to producemultivalent binding fragments, for example, by incorporation of acysteine residue that can form an intermolecular crosslink between twosuch modified fragments as described for variable heavy chain domains inWilliams et al., supra (1989). A CDR fragment having binding activitycan also be produced, for example, by expressing the CDR domain suchthat it is conformationaly constrained. Expression of a conformationalyconstrained CDR formed by addition of two cysteines capable of forming adisulfide bridge and having specificity for the antigen of the parentantibody is described, for example, in Ditzel et al., The J. Immunol.157:739-749 (1996). An antibody fragment of the invention can bemodified by any of a variety of additions, deletions or substitutions ofamino acids or other moieties such as labels as described herein.

The invention further provides a method of reducing neoplastic cellproliferation by administering to the cell an effective amount of ahuman monoclonal antibody or functional fragment produced by a cell lineselected from the group consisting of H1140, H2420 and H935. Alsoprovided is a method of reducing neoplastic cell proliferation byadministering to the cell an effective amount of a human monoclonalantibody or functional fragment having at least one CDR withsubstantially the amino acid sequence of a CDR of a sequence selectedfrom the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18,20, 12, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44. A humanmonoclonal antibody or functional fragment used for reducing neoplasticcell proliferation can further include a label, such as a cytotoxic orcytostatic agent, and can be combined with a pharmaceutical carrier.

An effective amount of an antibody or functional fragment thereof forreducing neoplastic cell proliferation is known or can readily bedetermined by one skilled in the art using in vitro methods or credibleanimal models. In vitro methods can include, for example, determining aneffective amount of a composition for reducing neoplastic cell growth orneoplastic cell mecastasis. A neoplastic cell used in an in vitro methodfor assaying reduction in growth or metastasis can be, for example, atumor cell line or an ex vivo culture of a tumor. The cell line or tumorcan be, for example, of breast, lung or ovarian tissue in origin. Aneffective amount for inhibiting neoplastic cell growth can be, forexample, an effective amount for inhibiting DNA synthesis, inhibitingcell division, inducing apoptosis, inducing non-apoptotic killing, orinhibiting angiogenesis. An effective amount for inhibiting metastasisof a neoplastic cell can be, for example, an amount effective forinhibiting cell motility, cell migration, cell attachment, cell invasionor cell proliferation.

An effective amount of an antibody or functional fragment thereof forreducing neoplastic cell proliferation can also be determined fromxenografts of human tumors in rodents. The rodent can be, for example, arat or a mouse. The mouse can be, for example, normal orimmunocompromised. An immunocompromised mouse can be, for example, anude mouse or a scid mouse. Such species are well known in the art andcan be obtained from commercial sources. Human cancer cells can beintroduced into an animal by a number of routes, includingsubcutaneously, intraveneously and intraperitoneally. Followingestablishment of a tumor, the animals can be treated with differentdoses of a molecule of the invention, and tumor mass or volume can bedetermined. Efficacy can be measured as partial or complete regressionof the tumor. An effective dose for treating cancer results in morepartial and complete regressions of tumors.

An effective amount of a molecule of the invention can be determined byone skilled in the art and will depend on such factors as age, bodyweight, sex and medical condition of the individual, and the particularroute of administration of the therapeutic agent. Useful routes ofadministration of a composition of the invention for treating cancerinclude, for example, intramuscular, intratumoral, intravascular,intraperitoneal, subcutaneous or intranasal routes.

The efficacy of a particular treatment in cancer patients can bedetermined by one skilled in the art. For example, in vivo or in vitrodiagnostic methods, such as those described below, can be used todetermine that a tumor has regressed or been eliminated followingtreatment. Additionally, normal prognostic indicators, such as survivaland increased quality of life for the cancer patient, can be used.

The invention provides a method of detecting neoplastic cells bycontacting a sample with a human monoclonal antibody or functionalfragment produced by a cell line selected from the group consisting ofH1140, H2420 and H935, and detecting the specific binding of the humanmonoclonal antibody or functional fragment to the sample, wherein thepresence or increased level compared to a normal cell of the humanmonoclonal antibody or functional fragment indicates the presence of orpredisposition to cancer. The invention also provides a method ofdetecting neoplastic cells by contacting a sample with a humanmonoclonal antibody or functional fragment having substantially theamino acid sequence of a CDR of a sequence selected from the groupconsisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42 and 44, and detecting the specificbinding of the human monoclonal antibody or functional fragment to thesample, wherein the presence or increased level compared to a normalcell of the human monoclonal antibody or functional fragment indicatesthe presence of or predisposition to cancer.

As used herein, the term “sample” is intended to mean any biologicalfluid, cell, tissue, organ or portion thereof, that includes orpotentially includes neoplastic cell. A biological fluid can be, forexample, blood or lymph. A tissue can be, for example, breast, ovary, orlung. The sample can be an in vivo or in vitro sample. An in vitrosample can be, for example, a histologic section, a specimen obtained bybiopsy, or cells that are placed in or adapted to tissue culture. Asample can be prepared by methods known in the art suitable for theparticular format of the detection method.

A sample can be contacted with a human monoclonal antibody or functionalfragment of the invention and specific binding of the human monoclonalantibody to the sample can be detected. Such contacting can be in vivoor in vitro, as determined by one skilled in the art depending on theformat of the detection method used. Specific binding of the humanmonoclonal antibody to the sample can be determined by immunoassays asdescribed above. Such immunoassays include, for example, both in vivoand in vitro immunoassays. In vivo immunoassays include, for example,radio imaging. Such a method involves contacting a sample within anindividual with a monoclonal antibody of the invention, and detectingspecific binding by, for example, radiographic imaging. In vitroimmunoassays include both qualitative and quantitative assays, such as,for example, immunohistochemistry, immunofluorescence, radioimmunoassay,FACS analysis, immunoblotting, immunoprecipitation and ELISA analysis,as described above.

The determination that neoplastic cells are present can be made bydetermining that a specifically bound human monoclonal antibody orfunctional fragment of the invention is present or is at an increasedlevel compared to a normal cell. As described above, one skilled in theart would be able to determine an appropriate normal cell to use forcomparison with a particular type of neoplastic cell.

The invention further provides a substantially pure human tumor-specificantigen. The term “tumor-specific antigen” is intended to mean anantigen which is preferentially expressed by human tumor cells. The term“preferentially expressed by human tumor cells” is intended to mean thata tumor-specific antigen is expressed by human tumor cells and isexpressed at a substantially lower level by normal human cells. Suchtumor cells that express a tumor-specific antigen of the invention canbe, for example, of breast, lung or ovarian tissue origin.

A human tumor-specific antigen of the invention is specifically reactivewith a human monoclonal antibody of the invention. Such an antigen canspecifically react with, for example, a human monoclonal antibody orfunctional fragment produced by LH11238, LH13, H1140, H2420 or H935hybridoma cell lines. Such an antigen can also specifically react with ahuman monoclonal antibody or functional fragment having at least one CDRwith substantially the amino acid sequence of a CDR of a sequenceselected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 44 or with ahuman monoclonal antibody or functional fragment having at least one CDRwith substantially the amino acid sequence of a CDR of SEQ ID NO:6 orSEQ ID NO:8. The antigen reactive with LH11238 human monoclonal antibodyor variants of fragments thereof is a protein present on the cellsurface and in lysosomal compartments of breast and ovarian cancer cellsas compared to normal human fibroblasts, peripheral blood lymphocytes,melanoma cells or lung carcinoma cells. The antigen reactive with LH13human monoclonal antibody is a secreted glycoprotein produced by breast,lung and ovarian cancer cells as compared to normal fibroblasts ormelanoma cells. Further properties of LH11238 and LH13 antigens aredescribed in the Examples.

Human tumor-specific antigens of the invention can beneficially be usedfor the treatment and diagnosis of cancer. For example, such antigenscan be used to generate additional binding agents that specifically bindthe human tumor-specific antigen for use in therapeutic and diagnosticprocedures. Such binding agents can, for example, inhibit or stimulatethe function of a tumor-specific antigen, or modulate the immune system,such that a neoplascic cell is growth-arrested or killed. Such bindingagents can also be conjugated to a label, such as, for example, acytotoxic or cytostatic agent, that causes the death or arrest of thetumor cells. Useful agents that bind to a tumor-specific antigen of theinvention include, for example, ligands, receptor antagonists andantibodies.

A substantially pure tumor-specific antigen can also be used in thetreatment of cancer by vaccinating a patient having cancer, or at riskof developing cancer with an effective amount of the antigen. Followingvaccination, the immune system of the individual will be able toprevent, reduce the proliferation of, or, eliminate neoplastic cellsexpressing such an antigen.

A substantially pure tumor-specific antigen of the invention can also bebeneficially used in methods for detecting binding of a tumor-specificbinding agent, such as a human monoclonal antibody or functional.fragment of the invention. For example, such an antigen can be used inan immunoassay such as competitive ELISA.

An appropriate starting material for isolating a substantially pureantigen of the invention can be identified by, for example, screening apanel of human tumor cells, using immunoaffinity procedures well knownin the art. For example, as described in Example II, ELISA analysis canbe used to determine a useful cell source of antigen for subsequentpurification. A useful cell source of antigen can be, for example,breast, ovarian, or lung carcinoma. A particularly useful startingmaterial for the purification of a tumor-specific antigen is the H3396breast carcinoma cell line.

An appropriate method for isolating a substantially pure antigen dependson the cellular localization of the antigen. For example, an antigen ofthe invention can be predominantly expressed in the secreted medium,cell surface membrane, vesicular membranes, cytoplasm, or nucleus of theneoplastic cell. Cellular localization of an antigen can be determinedby, for example, immunoassays well known in the art. For example, asdescribed in Examples IV and VI, indirect immunofluoresdence and ELISAanalysis can be used to establish the predominant localization of anantigen.

Purification of an antigen can be monitored by immunoaffinity proceduresknown in the art. Particularly useful methods of monitoring purificationinclude, for example, ELISA analysis and immunoblotting.

Tumor-specific antigen s can be purified from a particular source bybiochemical procedures well known in the art. For example, purificationcan include centrifugation, chromatographic methods, electrophoreticmethods and immunoaffinity methods, and can be chosen by one skilled inthe art depending on the characteristics of a particular antigen.Centrifugation procedures can be used to concentrate or enrich for asubcellular fraction which contains an abundant amount of the antigen.Such subcellular fractionation procedures are well known in the art.Chromatographic methods are also well known in the art, and includemethods of separating an antigen from contaminants based on its size ordifferential affinity for particular resins. Such resins can include,for example, size exclusion resins, ion exchange resins, and lectincolumns. As described in Example VI, a particularly useful resin forpurification of an antigen of the invention is Q SEPHAROSE FAST FLOW®agarose resin. Electrophoretic methods are also well known in the art,and include one and two-dimensional electrophoresis through, forexample, acrylamide or agarose gels. Immunoaffinity procedures are alsowell known in the art and include compounds conjugated to a humanmonoclonal antibody of the invention. Useful compounds for conjugatingan antibody for immunoaffinity purification of a tumor-specific antigeninclude chromatographic resins and Protein A.

Thus, following well-known biochemical procedures, one skilled in theart can readily isolate a substantially pure tumor-specific antigen ofthe invention for use in therapeutic and diagnostic procedures.

Substantially purified tumor-specific antigens of the invention can alsobe prepared from nucleic acids encoding tumor-specific antigens of theinvention by recombinant methods known to those skilled in the art.

The invention provides isolated nucleic acids encoding humantumor-specific antigens. Nucleic acids encoding human tumor-specificantigens can be isolated by methods known to those skilled in the art.Such methods include, for example, using monoclonal antibodies of theinvention to screen an expression library. Other methods include, forexample, screening a cDNA or genomic library using degenerateoligonucleotides as hybridization probes. The sequence of such adegenerate oligonucleotide can be determined by microsequencing anisolated tumor-specific antigen of the invention or fragment thereof.

Other methods known to those skilled in the art for producing a nucleicacid of a tumor-specific antigen include, for example, the polymerasechain reaction (PCR), using degenerate oligonucleotide primers obtainedfrom amino acid sequence of a tumor-specific antigen of the invention.Desired sequences can be amplified exponentially starting from as littleas a single gene copy by means of PCR.

The above described methods are known to those skilled in the art andare described, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1992) andthe various references cited therein and in Ansubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989); and in Harlow et al., Antibodies; A Laboratory Manual, ColdSpring Harbor Laboratory, New York (1989). These references and thepublications cited therein are hereby expressly incorporated byreference.

It is understood that modifications which do not substantially affectthe activity of the various embodiments of this invention are alsoincluded within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Production of Tumor-Specific Human Monoclonal Antibodies

This Example shows production of hybridomas that secrete humanmonoclonal antibodies specifically reactive with tumor cells. Aprocedure for immunizing normal lymphocytes in vitro with tumor cells orcell membranes prior to immortalization is described. This procedureallowed enrichment of lymphocytes producing novel tumor-reactive humanmonoclonal antibodies. These human monoclonal antibodies will be usefulfor cancer immunotherapeutic and immunodiagnostic procedures.

Lymphocytes were prepared as follows. Spleen tissue was isolated fromaccident victims, fragmented, and forced through a no. 50 mesh wirescreen (Bellco, Vineland, N.J.). The cells were collected bycentrifugation at 250×g for 10 min and RBC were removed by ammoniumchloride lysis. The remaining cells were washed, resuspended in freezingmedium (40% RPMI, 50% FCS and 10% DMSO) at a concentration of 100 to300×10⁶ cells/ml, frozen in 1.5-ml aliquots, and stored in liquidnitrogen. Both adherent cells and lymphocytes were isolated by thisprocedure.

A mixed lymphocyte reaction (MLR) was then established as follows.Frozen single-cell splenocyte preparations (described above) from twodifferent donors were thawed by gentle shaking at 37° C., washed twicewith RPMI, and collected by centrifugation for 10 min at 250×g. Afterthe second wash, 3×10⁶ splenocytes from each of the sources werecombined in 2 ml of RPMI containing 1.5 mM HEPES, pH 7.4 (FisherScientific), 10% FBS (HyClone), 2 mM L-glutamine, non-essential aminoacids, 1 mM sodium pyruvate, and 100 μg/ml gentamicin sulfate and placedin a 24-well tissue culture dish. This MLR, together with antigenicstimulation (described below), endogenously produced the requiredlymphokines.

MLR cultures were then stimulated with either mitomycin-treated H3396tumor cells, plasma membrane preparations from H3396 cells, orparaformaldehyde-fixed H3922 tumor cells. H3922 and H3396 are culturedcell lines established from metastases of human breast adenocarcinoma,which were explanted and maintained in culture. Each cell line wasderived from a different explant.

Mitomycin-treated H3396 cells were prepared as follows. H3396 cells wereplated in 24-well culture dishes, grown to confluency, and treated for12-15 h with 0.1 μg/ml mitomycin C. This concentration arrested celldivision in the tumor cell lines for approximately seven days. Followingthe 12-15 h incubation, the mitomycin C-containing media was removed andthe cells were washed three times with 2 ml of phosphate buffered saline(PBS).

Paraformaldeyde-fixed H2922 tumor cells were prepared by incubatingcells for 15 min at 25° C. in 2% paraformaldehyde in PBS after removingthe culture medium. The cells were then washed four times with PBS priorto use.

Plasma membranes from H3396 cells were isolated as follows. Tenconfluent 150-mm dishes of H3396 cells were each rinsed twice with 10 mlof ice-cold (Tris-buffered saline) TBS and harvested in 2 ml/150-mm dishof (Tris saline) TS containing 1 μg/ml leupeptin, 1 μg/ml pepstatin, and2 mM AEBSF. The cells were broken in a 15 ml Dounce homogenizer with 40strokes using a type A pestle. The lysate was centrifuged at 800×g at 4°C. for 5 min to remove unbroken cells and nuclei. The supernatant wassaved and the pellet was resuspended in 0.5 volumes of TS buffer,homogenized and centrifuged at 800×g. The supernatant was combined withthe first supernatant and centrifuged at 10,000×g at 4° C. for 2 h. Thesupernatant was discarded and the pellet was resuspended in 2 ml ofwater. A 2 ml Dounce and type B pestle was used to resuspend themembrane pellet. Phase separation of membranes in a 6.4% polymer systemwas performed on ice by mixing 2.56 ml of 20% dextran, 1.28 ml of 40%polyethylene glycol, 0.20 ml of 0.2 M potassium phosphate, pH 7.2, 0.8ml of 1 M sucrose, and 2.16 ml of water. To this mixture 1 ml ofmembranes were added and the tube was inverted 20 times end over end.The phases were separated by centrifugation at 800×g at 4° C. for 5 min.The upper phase was drawn off and mixed with the lower phase recoveredfrom a blank (water) sample. Likewise, the lower phase, including thematerial at the interface, was mixed with the upper phase recovered froma blank sample. Both phases were mixed, inverted 20 times end over end,and separated by centrifugation at 800×g at 4° C. for 5 min. Thematerial recovered from the upper phase of both samples was combined,the volume was adjusted to 21 ml with TBS, and the membranes werecollected by centrifugation at 100,000×g at 4° C. for 2 h. Thesupernatant was discarded and the pellet was resuspended with a 2 mlDounce homogenizer with a P pestle in 1-2 ml of TS buffer containing theprotease inhibitors. The membranes were stored at 4° C. for 12 h, atwhich time the insoluble material was separated from the supernatant bycentrifugation at 800×g at 4° C. for 10 min. The pellet was suspended asecond time in 1-2 ml of TS buffer containing protease inhibitors andstored as a suspension at 4° C. The supernatant is referred to asfraction 1 while the resuspended particulate material is referred to asfraction 2. Plasma membranes in fraction 1 were enriched greater than10-fold as determined by measuring 5′-nucleotidase or phosphodiesteraseactivity. The preparations had minimal succinate-dependent cytochrome Creductase (mitochondria), or NADPH-dependent cytochrome C reductase(endoplasmic reticulum) activity.

MLR cultures were immunized in vitro by incubation for three days withone of the following: monolayers of mitomycin C-treated H3396 cells,monolayers of paraformalehyde-fixed H3922 cells, 5 mg of plasma membranefraction 1 from H3396 cells, or with 10 mg of plasma membrane fraction 2from H3396 cells.

In vitro immunized lymphocytes were immortalized by either of twoalternative methods. In the first method, lymphocytes were fused withK6H6/B5 heteromyeloma cells. K6H6/B5 cells were maintained in spinnerculture in a 1:1 mixture of RPMI and Iscoves's modification of DMEM,supplemented with 10% FCS, 1% nonessential amino acids, 2 mM glutamine,and 1 mM sodium pyruvate. Approximately 4×10⁶ lymphocytes were combinedwith 2×10⁶ log phase K6H6/B5 cells and washed twice with RPMI. One ml ofa mixture of 35% polyethylene glycol (approximate m.w. 1450) and 7.5%DMSO in RPMI was added over 1 min, then gradually diluted with RPMI to 4ml and then diluted with RPMI supplemented with 10% FCS to 16 ml. Thelymphocyte concentration was then adjusted to 5×10⁴ cells/ml, and 0.2 mlof cells were seeded in 96-well cell culture dishes in hybridoma medium(RPMI supplemented with 10% FCS, 1% nonessential amino acids, 2 mMglutamine, 1 mM sodium pyruvate, 15 mM HEPES, pH 7.4 and 0.1 mg/mlgentamycin) containing HAT (13.6 μg/ml hypoxanthine, 3.8 μg/mlthymidine, 1 μg/ml azaserine) and 1.0 μM ouabain, to select againstunfused cells.

Hybridomas were screened for reactivity as described below, and clonesof interest were expanded. Using this one-step immortalization method,10-50 hybridomas were produced per 10⁶ lymphocytes fused; however, only5% of the interesting clones were stable through three rounds of cellculturing. Hybridoma clones H1140, H2420 and H935 were obtained by thisimmortalization procedure.

In an effort to improve clonal stablility, a second, two-step method forimmortalizing in vitro immunized lymphocytes was evaluated, involvingfirst transforming cells with EBV and then fusing clones of interestwith K6H6/B5 heteromyeloma cells. Equal volumes of lymphocytes (8×10⁴cells/ml) and EBV-transformed 1A2/C7 cells (2×10⁶ cells/ml) werecombined and the total volume was doubled by the addition of hybridomamedium. Hypoxanthine, thymidine and azaserine were added such that theirfinal concentrations were 13.6 μg/ml, 3.8 μg/ml, and 1 μg/ml,respectively. Two hundred microliters of the mixture of cells was platedper well in 96-well cell culture dishes to yield a final total of 4000lymphocytes and 1×10z,999 1A2/C7 cells per well. The cells were fed withhybridoma medium-HAT every three days and assayed for antibodyproduction (described below) after 2 weeks when colonies of cells werevisible.

Transformation of lymphocytes with EBV generated a higher percentage ofantibody-secreting clones than was obtained from fusions of lymphocyteswith the heteromyeloma line (50-100 antibody-secreting clones per 10⁶lymphocytes). However, lymphoblastoid clones generally secreted lowerlevels of antibody (less than 10 μg/ml) and, as was observed with thehybridomas, displayed poor long-term stability (approximately 5% of theinitial clones of interest were stable through three rounds ofexpansion). To address these limitations, tumor-reactive lymphoblastoidclones were fused with K6H6/B5 cells as described above, prior tomultiple rounds of expansion.

Formation of hybridomas from lymphoblastoid cells improved the stabilitysuch that 40% of the initial is lymphoblastoid clones were stablethrough three rounds of expansion following fusion. The combination ofEBV transformation followed by fusion with a heteromyeloma cell lineresulted in a higher frequency of immortalization of relevantlymphocytes than was achieved utilizing either approach alone. Inaddition, these clones generally secreted greater than 20 μg/ml ofantibody. Hybridoma clones LH11238 and LH13 were produced by thistwo-step immortalization method.

The immunization and immortalization conditions used to producehybridotna cell lines H1140, H2420, H935 LH11236 and LH13 are summarizedin Table 2. TABLE 2 Immunization and immortalization conditions used togenerate hybridoma cell lines producing tumor- specfific humanmonoclonal antibodies. Hybridoma Immunization Immortalization H1140 5 mgH3396 plasma Fusion with K6H6/B5 membrane fraction 1 cells H2420 5 mgH3396 plasma Fusion with K6H6/B5 membrane fraction 1 cells H935confluent monolayer of Fusion with K6H6/B5 mitomycin C-treated cellsH3396 cells LH11238 10 mg H3396 plasma EBV transformation membranefraction 2 and fusion with K6H6/B5 cells LH13 monolayer of 4 × 10⁴ EBVtransformation paraformaldehyde-fixed and fusion with H3922 cellsK6H6/B5 cells

Culture supernatants from immortalized lymphocytes were initiallyscreened for reactivity against monolayers of live primary tumor cellsusing an ELISA assay. This ensured that reactive antibodies recognizedsurface antigens, and also avoided artifacts associated with screeningfixed cells. Tumor cells were plated in 96-well cell culture dishes at acell density sufficient to produce 90-95% confluent monolayers 12-24 hlater. Just prior to use the media was removed and 50 μl of supernatantfrom either hybridomas or EBV transformed cells was added to the wellsand incubated at 4° C. for 2 h. Control wells (background) wereincubated with 50 μl of fresh hybridoma media.

Following incubation, supernatants were aspirated and-the cells weregently rinsed three times with 200 μl of PBS. The cells were thenincubated for 1-2 h with 50 μl of goat anti-human 1 g (H+L) alkalinephosphatase conjugate which had been diluted 1000-fold in 1% BSA-PBS.The detection antibody was aspirated and the cells were gently rinsedfour times as described above. The plates were developed for 1 h at 25°C. by the addition of 50 μl of 10 mM phenolphthalein mohophosphate in0.2 M 2-amino-2-methyl-1-propanol, 0.5 M Tris, pH 10.2 with 0.1% sodiumazide. The reaction was terminated by the addition of 50 μl of 30 mMTris, pH 10.2, with 15 mM EDTA and the absorbance at 560 nm wasdetermined. The background value for this assay was generallyA560<0.060. Clones with absorbances 4-fold or greater above backgroundwere selected for further characterization.

Antibodies that displayed reactivity with H3396 cells were also analyzedfor binding to fixed HF235 normal human fibroblasts. HF235 cells werefixed 15 min at 25° C. in 2% paraformaldehyde in PBS after removing theculture medium. The cells were then washed four times with PBS prior touse. The background value for the ELISA with fixed fibroblasts wasgenerally A560<0.080. Minimal reactivity with the fibroblasts wasdefined as binding less than 2-fold above background.

In order to determine the isotype of the antibodies produced by thehybridomas, microtiter plates were coated with 0.5 μg/ml either goatanti-human IgM or goat anti-human IgG. Antibody binding was detectedwith goat anti-human Ig alkaline phosphate conjugate. Development of theassay was terminated when supernatant yielded a positive signal with oneof the capture antibodies. Likewise, the light chain class wasdetermined by capturing the antibody with the appropriate heavy chainreagent and detecting with goat anti-human γ or κ chain-specificalkaline phosphatase conjugate.

Immunoglobulin quantitation was performed similarly, except thatsupernatants containing unknown quantities of Igs were serially diluteduntil reactivity was undetectable. Ig concentrations were calculatedfrom values of dilutions that fell within the linear range of thestandard curve, defined by standard samples of purified polyclonal humanIgM, used in the range of 0.01 to 2.0 μg/ml.

Immunoglobulins were precipitated from LH11238 hybridoma supernatant bycentrifugation at 800×g for 10 min and clarification by filtrationthrough a 0.45. μm cellulose acetate filter. The supernatant was placedin dialysis tubing (M_(r) cut-off 12-14 kDa) and concentrated two- tofour-fold by coating the tubing with Aquacide (Calbiochem) and placingit at 4° C. for 6-8 h. Excess Aquacide was removed, the dialysis bagrinsed, and the sample was dialyzed 12-24 h versus multiple changes ofice-cold distilled water. The precipitated antibody was collected bycentrifugation at 10,000×g for 30 min. The supernatant was removed andthe pellets were resuspended in a minimal volume of warm 10-foldconcentrated PBS. Following solubilization of most of the pellet, thebuffer concentration was adjusted to PBS and the insoluble material wasremoved by centrifugation at 10,000×g for 30 min. Antibodies H1140,H2420 and H935 could similarly be precipitated in low ionic strengthbuffer.

The LH13 antibody was not precipitated in low ionic strength buffer.Therefore, in order to obtain concentrated antibody the volume of theclarified supernatant was reduced with an Amicon apparatus utilizing aYM-30 membrane. The antibody was concentrated greater than 50-fold bythis approach.

Five percent of the hybridomas screened (372/7216) bound tumor surfaceantigens greater than four-fold above background. Of these 372 clones,55 (15%) produced antibodies that did not bind fibroblastssignificantly. Although numerous antibody-secreting hybridomas wereisolated from control MLRs that had not been incubated with tumor cellsor membrane fractions, none of these antibodies displayed preferentialtumor reactivity. Approximately 20% (11/55) of hybridomas producingantibodies that displayed tumor specificity were stable through multipleexpansions in culture. Five of these clones secreted greater than 20μg/ml antibody. The properties of these clones are summarized in Table2. All five antibodies were of the IgM isotype and displayed a range ofimmunoreactivities as determined by assaying on fixed monolayers ofH3396 cells. At least one antibody was generated from each of theculture conditions and immortalization procedures employed. The twoantibodies displaying the greatest immunoreactivity, LH13 and LH11238,were characterized further (below).

The properties of the tumor-specific human monoclonal antibodiesproduced by hybridoma cell lines H1140, H2420, H935 LH11238 and LH13 aresummarized in Table 3. TABLE 3 Characteristics of tumor-specific humanmonoclonal antibodies. Isotype/ Secretion Light chain Level Immuno-Precipi- Hybridoma class (μg/ml)^(a) Reactivity^(b) tation^(c) H1140IgM/λ 40 1.0 + H2420 IgM/λ 63 1.3 + H935 IgM/λ 36 1.4 + LH11238 IgM/κ 295.7 + LH13 IgM/λ 26 341 −^(a)Secretion level is typical value obtained from supernatant of aterminal culture^(b)Immunoreactivity (ΔA560/[(μg Ig) (min)]) determined on confluentparaformaldehyde-fixed H3396 monolayers and normalized toimmunoreactivity of H1140^(c)precipitation denotes ability (+) or inability (−) to preciptitateantibody in low ionic strength buffer

In summary, this example shows that an unstimulated human immunerepetoire (splenocytes from non-tumor-bearing patients) can be used togenerate human monoclonal antibodies reactive with novel tumor antigens.This was achieved through the stimulation of MLR cultures with eitherwhole tumor cells or membrane fractions derived from tumor cells. Thespecificity of the antigen stimulation was demonstrated by thegeneration of tumor-specific antibodies as well as by the absence oftumor-specific antibodies from cultures not treated with tumor cells ormembrane fractions.

EXAMPLE II Immunoreactivities of LH13 and LH11238 antibodies

This Example shows the immunoreactivities of LH13 and LH11238 antibodieswith a panel of human tumor cells.

In order to use LH13 and LH11238 antibodies for immunodiagnostic andimmunotherapeutic purposes, the range of tumor types expressing thecorresponding antigens needs to be identified. Tumor cell lines arerepresentative of the corresponding tumor type, and normal fibroblastsare representative of non-neoplastic tissues. The tumor cell lines werederived from melanomas, lung carcinomas, ovarian carcinomas and breastcarcinomas. The human monoclonal antibodies of the invention were testedfor immunoreactivity with both human cancer cell lines and normalfibroblasts.

H3396, H3464, H3477 and H3922 are cultured cell lines established frommetastases of human breast adenocarcinoma, which were explanted andmaintained in culture. H2981 and H2987 are cultured cell linesestablished from human lung carcinomas, which were explanted andmaintained in culture. H3639 and H3723 are culture cell linesestablished from human ovarian carcinomas, which were explanted andmaintained in culture. Each cell line was derived from a differentexplant.

Immunoreactivities were determined by the ELISA procedure describedabove, by incubating a broad range of antibody concentrations with fixedmonolayers of tumor cells. Antibody binding was measured with saturatingquantities of detect antibody. Antibody binding was measured as theΔA560/[(μg Ig)(min)] in the linear range of the assay. Theimmunoreactivities of tumor-specific human monoclonal antibodies againsta panel of tumor and normal cells are presented in Table 4. TABLE 4Immunoreactivity of human monoclonal antibodies LH13 and LH11228 againsttumor and normal cells. Immunoreactivity (ΔA560/[(μg Ig) (min)]) CellLine Description LH13 LH11238 HF285 normal fibroblast 0 0 H2669 melanoma0 0 H3774 melanoma 0 0 H3396 breast carcinoma 1.136 0.019 H3464 breastcarcinoma 0.507 0.022 H3477 breast carcinoma 0.046 0.008 H3922 breastcarcinoma  0^(a) 0 H2981 lung carcinoma 0.584 0 H2987 lung carcinoma 0 0H3639 ovarian carcinoma  0^(a) 0.011 H3723 ovarian carcinoma 2.602 0.026^(a)Exhibited substantial immunoreactivity upon permeabilization ofcells with 0.1% digitonin

As shown in Table 4, LH13 displayed a broad cross-reactivity on thepanel of tumor cells with high (H3723 and H3396), intermediate (H2981and H3464), and low (H3477) immunoreactivities. The LH13 antigen wasabsent or present at undetectable levels on intact normal fibroblasts(HF28S), melanomas (H2669 and H3774)”, and with several of the intactcarcinomas tested (H3922, H2987, and H3639). The lack of reactivity withH3922 cells was particularly surprising because the LH13 antibody wasisolated from lymphocytes which had been stimulated with H3922 cells. Toexamine this more closely each of the cell lines was permeabilized with0.1% digitonin and their reactivity with LH13 was re-examined. Two ofthe cell lines which were completely negative when intact cells wereassayed, H3922 and H3639, bound the LH13 antibody under conditions inwhich the antibody had access to intracellular compartments.Furthermore, H2981 cells, which had an intermediate LH13immunoreactivity when assayed under non-permeabilizing conditions werehighly reactive under permeabilizing conditions (immunoreactivitygreater than 7.0). The data obtained for immunoreactivities in thepresence and absence of 0.1% digitonin could not be compared directly,however, due to differential cell loss from the cell culture dishesduring the incubations.

LH11236 displayed a similar reactivity profile, though the magnitude ofits immunoreactivity was always much less than that of the LH13antibody. Reactivity was observed with intact H3396, H3464, H3477,H3639, and H3723. Other cells tested were negative, including thefibroblast and melanoma cell lines. Based on the immunoreactivitydeterminations made for each of the cell lines with LH13 and LH11238 itappears unlikely that these antibodies recognize the same epitope. Forinstance, LH13 displayed moderate immunoreactivity with the H2981 lungcarcinoma line while no binding of LH11238 to this cell line wasdetected. Likewise, LH11238 bound intact H3639 ovarian carcinoma cells,even though binding of LH13 could not be detected under identicalincubation conditions.

EXAMPLE III Binding Activity of LH13 and LH11238 Human MonoclonalAntibodies

This Example shows the binding activity of human monoclonal antibodiesLH13 and LH11238 with normal and tumor cells.

In order to determine the immunoreactivity of human monoclonalantibodies with live tumor cells and normal cells, flow cytometry (FACSanalysis) was used. H3464 breast tumor cells were chosen because theyexpressed both LH13 and LH11238 antigens by ELISA analysis and werereadily isolated as a single cell suspension. Normal peripheral bloodlymphocytes were also examined as representative of normal tissues. Facsanalysis additionally permits examination of heterogeneity of a cellpopulation with respect to antigen expression.

H3464 cells were removed from culture dishes with trypsin or Versene(EDTA), washed with PBS, resuspended at 2×10⁶ cells/ml in tumor media,and allowed to recover 2 h at 37° C. Antibodies were incubated at 5.0μg/ml with 1×10⁶ tumor-cells in 50 μl total volume for 30 min on ice.The cells were washed once with 1.0 ml of ice-cold PBS, incubated with 2μg/ml FITC-labeled goat anti-human IgM diluted in 1% BSA-PBS for 30 minon ice, and washed once more with 1.0 ml of ice-cold PBS. Antibodybinding to cells was analyzed with a Becton Dickinson FACSort.

Consistent with the ELISA results, H3464 cells incubated with LH11238and FITC-labeled antihuman IgM displayed a shifted staining patternrelative to an irrelevant control human IgM (FIG. 1A). Greater than 90%of the cells tested bound LH11238 antibody, though the broad range offluorescent intensities observed was consistent with heterogeneousexpression levels of the antigen on these tumor cells. Similar FACSstaining profiles were also observed with H935, H2420, and H1140.

Surprisingly, LH13 antibody, which was 23-fold more reactive thanLH11238 on H3464 cells as determined by ELISA (Table 4) displayed littleshift (FIG. 1B). To determine if the LH13 antigen was particularlysensitive to the proteolytic conditions used to isolate the tumor cellsfor analysis (trypsin), cells were allowed longer recovery periodsfollowing isolation or non-proteolytic cell isolation methods wereutilized, such as versene (EDTA) release. Neither of these approachesresulted in significantly greater staining of the tumor cells.

Facs analysis of normal human cells, such as peripheral bloodlymphocytes, was negative for each of the antibodies tested, consistentwith the ELISA results obtained with normal fibroblast cells.

EXAMPLE IV Characterization of LH11238 Antigen

This Example describes the subcellular characterization of the LH11238antigen.

Both the ELISA screening and the FACS analysis of live cells describedabove demonstrated the expression of LH11238 antigen on the plasmamembrane of tumor cells. However, these approaches did not examine thedistribution of the antigen on intracellular structures ofcarcinoma-derived cells. To examine the intracellular. localization ofLH11238 antigen, immunofluorescence analysis was used.

Monolayers of H3464 cells were seeded on 12-mm round coverslips (No. 1thickness, 0.06-0.13 mm thick) one day prior to use and were fixed with2% paraformaldehyde in PBS for 15 min at 25° C. The cells were rinsedtwice with PBS and incubated with 50 μl/ml antibody diluted either in 1%BSA-PBS (non-permeabilized) or in 1% BSA-PBS containing 0.1% digitonin(permeabilized) for 2 h at 4° C. The cells were rinsed twice with PBSand were then incubated in the dark with FITC-labeled goat anti-humanIgM diluted 1:500 in the same buffer as the primary antibody. The cellswere rinsed 4 times with PBS and the coverslips were mounted inFluoromount-G (Southern Biotechnology). Cells were visualized with anOlympus microscope equipped with epifluorescent optics and anOlympus-Splan 40× (NA 0.70) objective lens.

Non-permeabilized, paraformaldehye-fixed H3464 cells stained with theLH11238 antibody displayed surface staining consistent with localizationto the plasma membrane. Consistent with the FACS analysis, greater than70% of fixed H3464 cells bound LH11238. When H3464 cells werepermeabilized to allow access to intracellular structures, punctate,peri-nuclear staining was observed. LH11238 binding to H3464 cells wasspecific as control incubations with irrelevant, isotype andconcentration-matched human antibodies did not stain intact or 0.1%digitonin permeabilized cells. The punctate, peri-nuclear stainingobserved on permeabilized cells suggested localization of the antigen inthe lysosomes. To verify this, cells were stained with antibodies toCD63, a known lysosomal, glycoprotein. Incubation of H3464 cells withantibody to CD63 stained intracellular structures similar to thoselabeled with LH11238, consistent with a lysosomal localization ofintracellular LH11238.

Based on these results it was concluded that the LH11238 antigen ispresent both on the plasma membrane and in the lysosomes of H3464 cells.

EXAMPLE V

Internalization of LH11238 Antigen This Example shows that LH11238antigen is internalized from the plasma membrane to lysosomalcompartments.

Immunofluorescence experiments described above indicated that LH11238antigen was present on both the cell surface and in lysosomes of tumorcells. The dual localization could result from LH11238 antigen beingco-expressed in lysosomes and the cell surface, or from beinginternalized to endosomal/lysosomal compartments. In order to determinewhether LH11238 antigen was internalized, the procedure forimmunofluorescent localization was modified.

Monolayers of live H3464 cells were chilled on ice for 30-60 min tocompletely inhibit endocytosis. The cells were then washed, incubatedwith LH11238 antibody, washed again, and incubated with FITC-labeledgoat anti-human IgM antibody. The cells were maintained at 4° C.throughout all of the preceeding steps to ensure complete inhibition ofendocytosis. The cells were then shifted to 37° C. with pre-warmed cellculture media. At various intervals the cells were′ shifted back to 4°C. and fixed with 2% paraformaldehyde. Initially, diffuse surfacestaining was observed, identical to that observed with non-permeabilizedfixed cells. Following 10 min at 37° C., clustering of the LH11238antibody at multiple sites on the cell surface was observed. With longerincubation times, LH11238 first localized to a few sites on the cellsurface, and then began to internalize. These staining patterns were notobserved it cells were incubated with irrelevant primary antibodies,incubated with secondary antibody only, or if the cells were maintainedat 4° C. for the duration of the experiment. These results areconsistent with LH11238 binding and being internalized by H3464 cells.

In summary, the LH11238 antibody was first characterized as a surfaceantigen based on ELISA analysis of intact cells, FACS analysis of livecells, and immunolocalization using non-permeabilized cells. However,further immunolocalization studies with permeabilized cells demonstratedthat a portion of this antigen was also localized to a punctate,peri-nuclear compartment. Similar staining was observed with cellsincubated with anti-CD63 antibody, a lysosomal protein. Consistent withthe dual localization of the LH 11238 antigen on plasma membranes and inthe lysosomes, it was demonstrated that antibody bound to the surface oflive cells was internalized. The dual localization of tumor antigens tothe plasma membrane and lysosomes has been observed previously. Forinstance, the BR96 antibody binds the lysosomal-associated membraneprotein lamp-1 on the surface of intact tumor cells, even though lamp-1is normally an integral membrane protein predominantly located in thelysosomal compartment. In addition, the secretion of elevated levels ofsoluble lysosomal proteins, such as cathespins B, D, and L from tumorcells has also been documented. A portion of the secreted cathespin D isbound by the mannose 6-phosphate receptor and internalized. At present,it is unclear whether LH11238 is soluble or an integral membraneprotein. However, the antigen is found in the soluble fraction of aTX-114 phase separation of cell extracts, consistent with LH11238 beingmembrane-associated as opposed to an integral membrane protein.

EXAMPLE VI Characterization of LH13 Antigen

This example shows the localization of the LH13 antigen.

The LH13 antibody exhibited significant immunoreactivity when assayed byELISA on fixed monolayers of carcinoma-derived cell lines, as describedabove. However, FACS analysis suggested that LH13 bound only poorly tocell surfaces. Further ELISA analysis indicated that LH13 antigen waspredominantly present in a compartment that was only accessible toantibody when the cells were treated with 0.1% digitonin. To furtherexamine the expression of LH13 antigen in either subcellularcompartments or in the secreted medium, immunofluorescence analysis anddirect ELISA analysis were used.

To examine the subcellular localization of LH13 antigen,immunofluorescence was performed on paraformaldehyde-fixed H3464 cellsas described above. Incubation of LH13 antibody with unpermeabilizedH3464 cells (as described above using LH11238 antibody) resulted in weakstaining of the cell surface, consistent with the slight shift observedby FACS analysis. incubation of LH13 antibody with permeabilized H3464cells by immunofluorescence resulted in no detectable staining.Possibly, little LH13 is associated with intracellular structures inH3464 cells or intracellular forms of LH13 were not recognized by theantibody in this cell line.

These results could also indicate that LH13 antigen was predominantlysecreted. To observe secreted LH13 antigen, a constant amount of LH13antibody (0.01 μg/ml)was diluted into increasing amounts of culturemedia which had been removed from confluent monolayers of H3396 cells(conditioned media). Conditioned media was then assayed for binding tofixed H3396 monolayers. A modest reduction (approximately 15%) in thebinding of the LH13 antibody to the monolayer was observed withincreasing concentrations of conditioned media (FIG. 2A), consistentwith LH13 antigen being present in the media. No reduction in thebinding of the. LH13 antibody to the monolayer was observed withincreasing concentrations of conditioned media from H3922 cells.

To observe LH13 antigen in conditioned media of H3396 cells, a directELISA format was used. Conditioned media was incubated in a 96-well cellculture dish for 10-12 h at 4° C., removed, and the washed wells weresubsequently assayed for LH13 antibody binding. The antigen present inconditioned media obtained from H3396 cells bound to the culture dishand was readily detected (FIG. 2B). The specificity of antibody bindingwas demonstrated by incubating LH13 antibody with cell culture wellsthat had been pre-treated with: (1) conditioned media from a tumor lineexpressing undetectable quantities of LH13 (H3922), (2) tresh media(media), or (3) nothing (blank). None of these pre-incubation conditionsresulted in detectable LH13 binding to the well (FIG. 2B).

In summary, characterization of the LH13 antigen demonstrated that themajority of this antigen is found in conditioned media. Based on thisobservation, it was concluded that LH13 is secreted from tumor cells.

EXAMPLE VII Purification and Further Characterization of LH13 antigen

This example describes the purification and further characterization ofLH13 antigen.

As described above, the LH13 antigen is secreted from breast tumor cellsand displays a surprisingly high affinity for cell culture dishes. Theseobservations suggested that LH13 antigen could be readily purified fromconditioned H3396 media using its binding to culture dishes as a meansof monitoring its purification. Once the LH13 antigen is purified, itssusceptibility to various reagents could readily be determined tofurther characterize its properties.

As a first step in enriching the LH13 antigen, the H3396 cell line wasadapted to grow in serum-free media supplemented with minimal protein[Iscove's media supplemented with TCH (Celox, defined serumreplacement), 2 mM L-glutamine, non-essential amino acids, and 1 mMsodium pyruvate]. Cells grown under these conditions secreted comparablelevels of the antigen as grown in the presence of serum. Conditionedmedia was collected, pooled, and 250 ml was diluted to 1 liter withwater to reduce the ionic strength. The diluted media was applied at 3ml/min to a 11 cm×1.5 cm column of Q SEPHAROSE FAST FLOW agarose resinwhich had been equilibrated with 0.25-fold concentrated (0.25×) PBS. Themajority of the antigen bound to the column as demonstrated by the lackof reactivity of the flow-through fraction. After washing the columnwith 200 ml of 0.25× PBS, protein was eluted at a flow rate of 6 ml/minwith a 90 min linear gradient from 100% 0.25× PBS to 70% 0.5 M NaCl in0.25× PBS.

Column fractions were assayed for protein content with the BCA proteinassay using BSA as the standard. Column fractions were assayed for LH13antigen as follows. Each fraction was diluted 10-fold into water and 50μl/well was transferred to a sterile 96-well cell culture dish for 12 hat 4° C. The plate was washed twice with PBS and incubated with 10 μg/mlLH13 antibody diluted in 1% BSA-PBS for 2 h at 37° C. The plate was thenwashed four times with PBS, incubated with goat anti-human 1 g alkalinephosphatase conjugate diluted 1000-fold into 1% BSA-PBS for 1 h at 25°C., and was developed as described above.

Most of the protein eluted between 150 mM and 250 mM NaCl (FIG. 3,closed circles), while the majority of the LH13 antigen eluted above 250mM NaCl (FIG. 3, open circles). The specific activity (ELISAsignal/[protein]) of the peak antigen fraction was 200-fold greater thanthe starting material. As a control, LH13 antigen was diluted with arange of NaCl concentrations (50 mM to 400 mM) to determine it the ionicstrength affected binding to the cell culture dishes. The range of NaClconcentrations employed for binding and eluting LH13 from the column didnot affect antigen binding to the cell culture dish. Therefore, thedirect ELISA accurately reflects the distribution of antigen in thevarious column fractions.

Column fractions were further resolved by electrophoresis on 4-20%SDS-polyacrylamide gradient gels. Coomassie blue staining revealed asingle major protein in the most reactive fractions, but Western blotsof the various column fractions failed to identify this as theLH13-reactive antigen.

To further characterize the LH13 antigen, its susceptibility to avariety of treatments was examined using the direct ELISA method. LH13antigen was coated on cell culture dishes, as described above, andincubated under a variety conditions. LH13 antigen was incubated with 5mg/ml trypsin at 37° C. for 30 min. Alternatively, LH13 antigen wasdenatured by treatment with 10% SDS +2% β-mercaptoethanol at 37° C. for30 min. prior to treatment with 2 U/ml sialidase (Oxford GlycoSystems),400 U/ml endoglycosidase-F/peptide-N-glycosidase F (Endo F, PNGaseF)(Oxford GlycoSystems), 60 mU/ml endo-α-N-acetylgalactosaminidase(0-glycanase)(Oxford GlycoSystems), or 2 U/ml sialidase plus 60 mU/mlendo-α-N-acetylgalactosaminidase (Sialidase, 0-glycanase) at 37° C. for24 h. The culture dishes were washed three times with PBS and assessedfor LH13 antibody binding as described above. The effect of eachtreatment was compared to control samples which were treated underidentical conditions with buffer alone (FIG. 4).

Treatment of LH13 antigen with 10% SDS +2% β-mercaptoethanol, sialidase,or endo-α-N-acetylgalactosaminidase (O-glycanase) had little effect onsubsequent binding of antibody. The resistance of the antigen totreatment with sialidase and O-glycanase was not affected by denaturingthe antigen prior to treatment nor by simultaneous treatment with bothglycosidases. Binding of antibody in the direct ELISA was reduced bytreatment with trypsin (79%, FIG. 4) or withendoglycosidase-F/peptide-N-glycosidase F (36%, not shown). The bindingof antibody to LH13 denatured with 10% SDS+2% β-mercaptoethanol prior totreatment with endoglycosidase-F/peptide-N-glycosidase F was furtherdiminished, reducing binding by 75% as compared to untreated samples(FIG. 4).

Due to the broad range of specificities ofendoglycosidase-F/peptide-N-glycosidase F it was not possible to reachspecific conclusions regarding the structure of the carbohydrate.However, binding of LH13 antibody was completely unaffected by treatmentwith sialidase, which removes N- or O-acyl, non-reducing terminal sialicacids. Treatment with endo-α-N-acetylgalactosaminidase, which removesGaβ1-3GalNAc associated with serine or threonine, or the combination ofsialidase and endo-α-N-acetylgalactosaminidase also did not affectantibody binding. Trypsin sensitivity of antibody binding to antigensuggested that the epitope is associated with a protein component.

In summary, LH13 antigen was purified more than 200-fold from H3396serum-free conditioned medium, using anion exchange chromatography.Preliminary studies indicated that purified LH13 antigen is excludedfrom a Superdex 75 gel filtration column, consistent with the antigenbeing larger than 70 kDa. Treatment of LH13 antigen bound to cellculture plates with a variety of reagents provided evidence that bothcarbohydrate and protein components are present, and are involved ineither binding of the epitope to LH13 antibody or binding LH13 antigento tissue culture plates. At present, it is not possible to distinguishbetween these two possibilities.

EXAMPLE VIII In vitro Antibody Maturation

This example demonstrates cloning of antibody variable regions,synthesis of Fab libraries containing variants of the cloned variableregions, and screening to identify variants with improved affinity.

DNA sequencing of LH13 and LH11238 demonstrated that the H and L chainsof both antibodies were highly homologous to human germline sequences.The LH11238 VH is identical to germline sequence DP-63 (VH4.21) whilethe LH13 VH is identical to DP-10/hv1051. ,(Vh1-69). Furthermore, theLH11238 VL is identical to germline sequence DPK21/humkv328h5 while theLH13 VL is highly homologous to DPL16/VL3.1, containing a singlenucleotide change at the V-J junction. The high degree of homologybetween the antibodies and germline sequences demonstrated that theantibodies probably did not undergo appreciable affinity maturation inthe cell cultures. In order to rapidly enhance the physical attributesof the LH13 and LH11238 antibodies through protein engineering, theantibodies were cloned into a bacterial expression system, as set forthbelow.

Antibody VH and VL variable regions were cloned as follows. Total RNAwas isolated from 5×10⁷ cells of each clone and fist strand cDNA wasprepared. The H chain variable regions were amplified by PCR using thefollowing set-of 5′ primers homologous to human signal sequences and a3′ primer (5′-AGACGAGGGGGAAAAGGGTT-3′, SEQ ID NO:47) corresponding tohuman CH1: ATGGAGTTTGGGCTGAGCTGG (SEQ ID NO:48),ATGGACTGGACCTGGAG(G/C)(A/T/G)TC (SEQ ID NO:49),ATGAA(A/G)CA(C/T)CTGTGGTTCTT (SEQ ID NO:50), ATGGGGTCAACCGCCATCCTC (SEQID NO:51), ATGGGATGGAGCTGTATCATC (SEQ ID NO:52), ATGTCTGTCTCCTTCCTCATC(SEQ ID NO:53), and ATG(A/G)AC(C/A)TACTTTGTT(G/C)C (SEQ ID NO:54). Thekappa light chain variable regions were amplified using a set of 5′primers encoding signal sequence(CT(C/T)CT(G/C)(G/T)(G/T)(G/C)CTCCTGCT(A/G)CTCTGG, SEQ ID NO:55 andCT(C/T)CT(G/C)(G/T)(G/T)(G/C)CT(C/G)CT(G/A)(C/G/A/T)T(A/G)CTCTGG, SEQ IDNO:56) and a 3′ primer corresponding to constant region amino acids117-122 (CATCAGATGGCCGGGAAGAT, SEQ ID NO:57). Similarly, the lambdalight chain variable regions were amplified using a set of 5′ primersencoding signal sequence(ATG(A/G)CCTG(C/G)(A/T)C(C/T)CCTCTC(C/T)T(C/T)CT(C/G)(A/T)(C/T)C, SEQ IDNO:58) and a 3′ primer corresponding to constant region amino acids115-124 (CTCCTCAGAGGAGGGCGGGAACAGAGTGAC, SEQ ID NO:59). Following PCRamplification, DNA fragments were purified o01 an agarose gel and clonedinto the pCR2.1 vector (Invitrogen, Carlsbad Calif.). Multiple clones ofeach sample were sequenced on both strands by the fluorescentdideoxynucleotide termination method using M13 forward and reverseprimers (Perkin-Elmer, Foster City, Calif.). A slash between nucleotidesat a particular position in a sequence is intended to indicate an equalmolar mixture of those nucleotides at that position.

In order to convert the IgMs to more useful therapeutic mAbs theisotypes were switched from IgM to IgG1 by grafting the H chain variableregions directly to a γ1l constant region. In vitro class switching wasdone to increase control over antibody effector functions and to exploitbetter characterized expression, production, and purificationmethodologies for therapeutic mAbs. The cloned VH and VL regions of theLH11238 antibody were cloned into the phage vector M13IX104CS (describedin Wu et al., J. Mol. Biol. 294:151-162 (1999) and Kristensson et al.,Vaccines 95, Cold Springs Harbor Laboratory Press, Cold Springs Harbor,N.Y. pp39-43 (1995) in frame to human IgG CH1 and kappa CL sequences byhybridization mutagenesis (as described in Rosok et al., J. Biol. Chem.271:22611-22618 (1996)). The LH13 antibody was cloned into the samevector in which the kappa CL had been replaced with lambda CL sequence.

In order to rapidly improve the affinity of the antibodies in vitro,while minimizing the potential of altering the epitope specificity,libraries of HCDR3 and LCDR3 variants closely related to the parentsequence were synthesized and screened. There are multiple mechanisms invivo by which diversity is introduced to a greater extent in HCDR3 andLCDR3 than in the other CDRs of the antibody, consistent with theseregions serving a key role in antigen recognition. Moreover, duringprevious in vitro affinity maturation of unrelated mAbs, the greatestnumber of beneficial mutations were typically located in HCDR3 andLCDR3. Therefore, the initial libraries were focused in the third CDR ofthe H and L chains and the variants altered from the parental sequenceby a single amino acid change.

Using the numbering system of Kabat et al. supra, the residues chosenfor mutagenesis of the LH13 CDRs were: Asn89 through Va197 in L chainCDR3 (LCDR3) and Glu95 through Tyr102 in H chain CDR3 (HCDR3) and theresidues chosen for mutagenesis of the LH11238 CDRs were: Gln89-Thr97 inLCDR3 and Glu95-Tyr102 in HCDR3. The initial libraries and thecombinatorial LH13 library based on the beneficial LCDR3 and HCDR3mutations were synthesized as described in Wu et al., Proc. Natl. Acad.Sci. USA 95:6037-6042 (1998) and are shown in Table 5. TABLE 5 SequenceHeavy Light Anti- Chain Chain body Library Clone CDR3 CDR3 LH13 HCDR3Wild- E D S S G W Y H Y type S97G     G S97T     T S97N     N LCDR3Wild- N S R D S S G N P V V type R91Y     Y R91F     F V97Y                    Y V97F                     F Combin- 4D5     T     Yatorial 4E2     T     Y               F 4H7     T     F               F4G11     F               F 3G4     Y               F LH11238 LCDR3 Wild-Q Q Y N N W P P Y T type Q89L L Q89G G Q89V V Q89F F Q89W W N93C       CN94C         C P95aF               F P95aR               R

The antibody variants in these focused libraries each contained a singlemutation, and all 20 amino acids were introduced at each residue of bothCDRS. Thus, the LH11238 libraries contained 228 (HCDR3) and 190 (LCDR3)distinct non-wild-type variants while the LH13 libraries contained 171(HCDR3) and 209 (LCDR3) distinct variants.

Fabs were expressed in E. coli XL1-blue cells (Stratagene) as describedin Wu et al, supra (1999) and Fabs were isolated from the periplasmicspace and quantitated as described in Watkins et al., Anal. Biochem.253:37-45 (1997). Soluble Fabs were screened in an ELISA format. Thebinding of soluble LH13 Fab to partially purified LH13 antigen wasdetected with goat anti-human lambda chain alkaline phosphataseconjugate. LH13 antigen was partially purified as described in ExampleVII. LH11238 Fab binding was determined using fixed H3396 tumor cells asdescribed in Watkins et al., supra (1997).

Bacterially expressed LH13 and LH11238 Fabs displayed relatively lowreactivity in the ELISA as shown in FIG. 5, (open circles) indicatingthat the antibodies had not undergone significant antibody maturation invivo. The results further are consistent with the observation that thebinding of 1 gM molecules is driven largely by multivalency. Screeningof the focused libraries identified multiple variants of each antibodythat contained single amino acid changes and displayed as much as100-fold greater binding than the wild-type antibodies as indicated bycomparison of open circles with open squares in FIGS. 5A and 5B.Beneficial mutations in the LH13 antibody were identified in HCDR3 andLCDR3. As shown in FIG. 5A, clone S97N (filled circles) and clone R91Y(open squares) had enhanced binding compared to the LH13 parent.Multiple beneficial mutations were also identified in LCDR3 of LH11238including clone N94C (filled squares), clone N93C (open triangles) andclone P95aR (filled circles) as shown in FIG. 5B.

Variants displaying enhanced binding were sequenced to identify themutations that resulted in higher affinity and are also listed in Table5. The LH13 variant displaying the highest binding activity (FIG. 5A,open squares) contained an R91Y mutation in LCDR3 and had a K_(d) ofabout 1.8 nM. The LH11238 mutant displaying the greatest increase inaffinity (FIG. 5B, open squares) was the result of a Q89W mutation inLCDR3 and had a K_(d) of about 11 nM. Limited screening of the librariesidentified seven LH13 variants and nine LH11238 variants that displayedgreater than 2-fold enhanced binding all of which are listed in Table 5.Beneficial mutations of LH13 CDRs were identified at S97 of HCDR3 andR91 and V97 of LCDR3 while the improved LH11238 variants containedbeneficial changes at Q89, N93, N94, and P95a of the LCDR3. Theseresults indicated that single amino acid changes in LCDR3 of LH13increased the affinity by greater than 50 fold while single amino acidchanges in LCDR3 of LH11238 increased the affinity greater than 35 fold.

A combinatorial library of the beneficial mutations identified in bothHCDR3 and LCDR3 of LH13 was synthesized to combine individual mutationsand further enhance antibody affinity. The library contained everypossible combination of the individual mutations or wild-type amino acidat each of the three CDR positions (S97 of HCDR3 and R91 and V97 ofLCDR3), resulting in a library that contained 36 distinct variants.Combinatorial clones displaying higher affinity, than the variantscontaining single amino acid changes were readily identified as shown bycomparison of combinatorial mutant 4H7 (filled squares) with LCDR3variant R91Y (open squares) or HCDR3 variant S97N (filled circles) inFIG. 5A. Combinatorial mutant 4H7 had a K_(d) of about 1.1 nM. DNAsequencing of the combinatorial clones demonstrated that all containedat least two mutations, as shown in Table 5, and two contained threemutations (clones 4E2 and 4H7). All five combinatorial clones displayedgreater binding activity than any of the variants containing a singlemutation.

This example demonstrates affinity maturation of LH13 accomplished bysynthesis of only 416 distinct protein variants in two steps. As setforth above, the first step consisted of screening 171 HCDR3 mutants and209 LCDR3 mutants followed by a second step in which 36 combinatorialvariants were screened. In contrast, total randomization of the threeCDR residues demonstrated to influence activity in this study, LCDR3S97, HCDR3 R91, and HCDR3 V9, would have required the expression of alibrary containing 19³, or 6,859, variants. Thus, the results describedhere demonstrate that stepwise improvement in affinity capturedadditivity of independent mutations and further provided an efficientmethod of affinity maturation.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. An isolated human monoclonal antibody or functional fragment thereofcomprising a light chain variable region and a heavy chain variableregion with wherein the light chain variable region comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 28, 30,32, 34, 36, 38, 40, 42 and 44, and wherein the heavy chain variableregion comprises an amino acid sequence of SEQ ID NO: 2, and whereinsaid antibody or functional fragment thereof binds a neoplastic cell orantigen thereof.
 2. The isolated human monoclonal antibody of claim 1,wherein said functional fragment is selected from the group consistingof Fv, Fab, Fab′, and F(ab′)₂.
 3. The isolated human monoclonal antibodyor functional fragment of claim 1, further comprising a label.
 4. Theisolated human monoclonal antibody or functional fragment of claim 3,wherein said label comprises a cytotoxic or cytostatic agent.
 5. Acomposition comprising an isolated human monoclonal antibody orfunctional fragment of claim 1, wherein the composition isphysiologically acceptable and used for the treatment of cancer.